Mice and ethics statement
Tug1tm1.1Vlcg-knockout mice have been described previously [20, 41]. To remove the loxP-flanked neomycin resistance gene included in the targeting construct, we crossed Tug tm1.1Vlcg mice to C57BL6/J mice and then to a cre-recombinase strain (B6.C-Tg(CMV-cre)1Cgn/J, The Jackson Laboratory, 006054). Mice free of both the neomycin-resistance and cre-recombinase genes were selected for colony expansion and subsequently backcrossed to C57BL/6J mice. The Tug1-knockout allele was maintained by heterozygous breeding, and mutant mice were identified by genotyping for loss of the Tug1 allele and gain of the lacZ cassette (Transnetyx, Inc.).
For allele-specific gene expression analyses, we generated Tug1BL6-KO/Cast-WT mice by crossing inbred Mus castaneus (Cast/EiJ) males (The Jackson Laboratory, 000928) with inbred heterozygote Tug1 females. The F1 hybrid male progeny (three wild-type Tug1BL6-WT/Cast-WT and four with a maternal Tug1-knockout allele Tug1BL6-KO/Cast-WT) were used for allele-specific expression studies.
To generate an inducible Tug1-overexpression mouse, tg(Tug1), we cloned Tug1 cDNA (see the “Sequences and primers” section) into a Tet-On vector (pTRE2). Full-length Tug1 (Ensembl ID: ENSMUST00000153313.2) was amplified from Riken cDNA clone E330021M17 (Source Bioscience) using specific primers containing MluI and EcoRV restriction sites (see the “Sequences and primers” section). After gel purification, we sub-cloned the amplicon using the MluI and EcoRV restriction sites into a modified Tet-On pTRE2pur vector (Clontech 631013) in which the bGlobin-intron was removed. We verified the absence of mutations from the cloned Tug1 cDNA by sequencing (see the “Sequences and primers” section). We injected this cassette into the pronucleus of C57BL/6J zygotes (Beth Israel Deaconess Medical Center Transgenic Core). Two male founder mice containing random integration of the tg(Tug1) cassette were identified by genotyping for the pTRE allele and individually mated to female C57BL/6J mice (The Jackson Laboratory, 000664) to expand the colonies. Next, we generated quadruple allele transgenic mice to test the functionality of the Tug1 RNA by the following strategy. We mated tg(Tug1) males to Tug1tm1.1Vlcg females and identified male progeny that were Tug1+/−; tg(Tug1). These mice were then mated to female rtTA mice (B6N.FVB(Cg)-Tg(CAG-rtTA3)4288Slowe/J mice (The Jackson Laboratory, 016532)), and we identified male progeny that were Tug1+/−; tg(Tug1), rtTA. Finally, we mated male Tug1+/−; tg(Tug1), rtTA mice to Tug1+/− females, and at the plug date, females were put on 625 mg/kg doxycycline-containing food (Envigo, TD.01306). We genotyped progeny from the above matings (Transnetyx, Inc.) and identified male progeny that were Tug1−/−; tg(Tug1), rtTA, and maintained these mice on the doxycycline diet until the experimental end point.
Cell lines and cell culture
We derived primary wild-type and Tug1−/− mouse embryonic fibroblasts (MEFs) from E14.5 littermates from timed Tug1+/− intercrosses as described [80]. We maintained MEFs as primary cultures in DMEM, 15% FBS, pen/strep, l-glutamine, and non-essential amino acids. We genotyped MEFs derived from each embryo and used only male Tug1−/− and wild-type littermate MEFs at passage 2 for all experiments. 3T3 (ATCC, CRL-1658), HeLa (ATCC, CRM-CCL-2), and BJ (ATCC, CRL-2522) cell lines were purchased from ATCC and cultured as recommended.
Whole-mount in situ hybridization
We generated an antisense riboprobe against Tug1 (see the “Sequences and primers” section) from plasmids containing full-length Tug1 cDNA (Ensembl id: ENSMUST00000153313.2) and performed in situ hybridization on a minimum of three C57BL6/J embryos per embryonic stage. For whole-mount staining, embryos were fixed in 4% paraformaldehyde for 18 h at 4 °C, followed by three washes in PBS for 10 min. We then dehydrated embryos through a graded series of 25%, 50%, and 75% methanol/0.85% NaCl incubations and then finally stored embryos in 100% methanol at − 20 °C. Embryos were then rehydrated through a graded series of 75%, 50%, 25%, and methanol/0.85% NaCl incubations and washed twice with PBS with 0.1% Tween-20 (PBST). Embryos were treated with 10 mg/mL proteinase K in PBST for 10 min (E8.0, E9.5) or 30 min (E10.5, E11.5, and E12.5). Samples were fixed in 4% paraformaldehyde/0.2% glutaraldehyde in PBST for 20 min at room temperature and washed twice with PBST. We then incubated samples in pre-hybridization solution for 1 h at 68 °C and then incubated samples in 500 ng/mL of Tug1 antisense or sense riboprobe at 68 °C for 16 h. Post-hybridization, samples were washed in stringency washes and incubated in 100 μg/mL RNaseA at 37 °C for 1 h. Samples were washed in 1× maleic acid buffer with 0.1% Tween-20 (MBST) and then incubated in Roche Blocking Reagent (Roche, #1096176) with 10% heat-inactivated sheep serum (Sigma, S2263) for 4 h at room temperature. An anti-digoxigenin antibody (Roche, 11093274910) was used at 1:5000 and incubated for 18 h at 4 °C. Samples were washed 8 times with MBST for 15 min, 5 times in MBST for 1 h, and then once in MBST for 16 h at 4 °C. Prior to developing, the samples were washed three times with NTMT (100 mM NaCl, 100 mM Tris-HCl (pH 9.5), 50 mM MgCl2, 0.1% Tween-20, 2 mM levamisole). The in situ hybridization signal was developed by adding BM Purple (Roche, 11442074001) for 4, 6, 8, and 12 h. After the colorimetric development, samples were fixed in 4% paraformaldehyde and cleared through a graded series of glycerol/1× PBS and stored in 80% glycerol. Imaging was performed on a Leica M216FA stereomicroscope (Leica Microsystems) equipped with a DFC300 FX digital imaging camera.
Tug1 single-molecule RNA FISH
We performed Tug1 single-molecule RNA FISH as described previously [81]. Briefly, 48 oligonucleotides labeled with Quasar 570 and Quasar 670 tiled across human/mouse Tug1 transcripts were designed with LGC Biosearch Technologies’ Stellaris probe designer (Stellaris Probe Designer version 4.2) and manufactured by LGC Biosearch Technologies.
Human foreskin fibroblasts (ATCC® CRL-2522™) and mouse 3T3 fibroblasts (ATCC, CRL-1658™) were seeded on glass coverslips previously coated with poly-l-lysine (10 μg/mL) diluted in PBS. Prior to hybridization, coverslips were washed twice with PBS, fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, and washed twice more with PBS. Coverslips were immersed in ice-cold 70% EtOH and incubated at 4 °C for a minimum of 1 h. We then washed the coverslips with 2 mL of Wash Buffer A (LGC Biosearch Technologies) at room temperature for 5 min. Next, we hybridized cells with 80 μL hybridization buffer (LGC Biosearch Technologies) containing Tug1 probes (1:100) overnight at 37 °C in a humid chamber. The following day, we washed the cells with 1 mL of Wash Buffer A for 30 min at 37 °C, followed by another wash with Wash Buffer A containing Hoechst DNA stain (1:1000, Thermo Fisher Scientific) for 30 min at 37 °C. Coverslips were washed with 1 mL of Wash Buffer B (LGC Biosearch Technologies) for 5 min at room temperature, mounted with ProlongGold (Life Technologies) on glass slides, and left to curate overnight at 4 °C before proceeding to image acquisition (see below).
Sperm counts and morphology
Tug1−/− (n = 8) and wild-type (n = 9) males between 8 and 41 weeks of age were sacrificed and weighed. We then dissected the entire male reproductive tract in phosphate-buffered saline (PBS). One testis was removed, weighed, and fixed in 4% paraformaldehyde (PFA) for histology (see below). Sperm were collected from one cauda epididymis by bisecting and suspending the tissue in a solution of Biggers-Whitten-Whittingham (BWW) sperm media at 37 °C. After a 15-min incubation, we used the collected sperm solutions to analyze sperm morphology and counts.
We characterized sperm morphology by fixing sperm in 2% PFA in PBS, mounting 20 μL of suspended sperm in Fluoromount-G media (Southern Biotech) on Superfrost glass slides (Thermo Fisher Scientific) and scanning each slide in a linear transect, recording the morphology as normal or abnormal for each sperm cell encountered (between 30 and 120 sperm). When abnormal, we also recorded the type of morphological defects: headless, head angle aberrant, head bent back to midpiece, debris on the head, debris on the hook, head misshapen, midpiece curled, midpiece kinked, midpiece stripped, debris on the midpiece, tailless, tail curled, tail kinked, broken tail, or multiple cells annealed together.
Sperm counts for each Tug1−/− (n = 7) and wild-type (n = 9) mice were determined using a Countess Automated Cell Counter according to the manufacturer’s protocol (Life Technologies, Carlsbad, CA). For the Tug1rescue experiment, sperm counts for control (WT and Tug1+/−) (n = 2), Tug1−/− (n = 2), and Tug1−/−; tg(Tug1); rtTA mice (n = 3) were determined by manual counts using a hemocytometer. For all analyses, statistical comparisons between Tug1−/− and wild type were performed using the two-tailed Wilcoxon rank-sum tests with an a = 0.05. The results for testis, sperm count, and morphological parameters are presented in Additional file 5: Table S1. All statistical comparisons of Tug1−/− versus wild type for relative testis size, sperm morphology, and sperm counts were performed using R (Wilcoxon rank-sum test and principal component analysis (PCA)).
lacZ and histological staining of male reproductive tissues
Expression of the knock-in lacZ reporter and histological staining for morphological analysis of male reproductive tissues was conducted on the testes and epididymis from Tug1−/− (n = 2) and wild-type (n = 2) mice. We fixed the testis and epididymis in 4% paraformaldehyde in PBS overnight at 4 °C and washed the tissues three times in PBS. For lacZ staining, we rinsed Tug1+/− and wild-type tissues three times at room temperature in PBS with 2 mM MgCl2, 0.01% deoxycholic acid, and 0.02% NP-40. We performed X-gal staining by incubating the tissues for up to 16 h at 37 °C in the same buffer supplemented with 5 mM potassium ferrocyanide and 1 mg/mL X-gal. The staining reaction was stopped by washing three times in PBS at room temperature, followed by 2 h post-fixation in 4% paraformaldehyde at 4 °C.
We then embedded the organs in paraffin, sectioned the organs at 6 μm thickness, and then mounted the sectioned samples onto glass microscope slides. The testis sections were additionally stained with Mayer’s hematoxylin, periodic acid, and Schiff’s reagent (VWR, 470302-348), and the epididymis sections were stained with eosin (VWR, 95057-848). Images were collected using a Zeiss AxioImager.A1 upright microscope or on an Axio Scan Z.1 (Zeiss).
RNA isolation and RNA-seq library preparation
We isolated total RNA from mouse tissues, mouse embryonic fibroblasts (MEFs), and blood cells using TRIzol (Invitrogen, 15596026) by chloroform extraction followed by spin-column purification (RNeasy mini or micro kit, Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were determined using a Nanodrop. We assessed RNA integrity on a Bioanalyzer (Agilent) using the RNA 6000 chip. High-quality RNA samples (RNA integrity number ≥ 8) were used for library preparation. We then constructed mRNA-seq libraries using the TruSeq RNA Sample Preparation Kit (Illumina) as previously described [82]. The libraries were prepared using 500 ng of total RNA as input and a 10-cycle PCR enrichment to minimize PCR artifacts. Prior to sequencing, we ran libraries on a Bioanalyzer DNA7500 chip to assess purity, fragment size, and concentration. Libraries free of adapter dimers and with a peak region area (220–500 bp) ≥ 80% of the total area were sequenced. We then sequenced individually barcoded samples in pools of 6, each pool including Tug1 mutant and wild-type samples, on the Illumina HiSeq platform using the rapid-full flow cell with the 101-bp paired-end reads sequencing protocol (Bauer Core, Harvard University FAS Center for System Biology).
RNA-seq and gene set enrichment analyses
We mapped sequencing reads to the reference mouse genome (GRCm38) by STAR [83] with the gene annotation obtained from GENCODE (vM16). We counted uniquely mapped reads for genes by featureCounts [84] and calculated transcripts per million (TPM) for genes to quantify the gene expression level after normalization of sequencing depth and gene length. Clustering of gene expression between tissues was done with Ward’s method using Jensen-Shannon divergence between tissues as the distance metric. The R package, Philentropy, was used for calculation of the Jensen-Shannon divergence [85].
We identified differentially expressed genes by comparing the mean read counts of biological replicates between the groups (wild-type vs. Tug1-/- and Tug1-/- vs. Tug1rescue) using the generalized linear model. Statistical significance was calculated with the assumption of the negative binomial distribution of the read counts and with the empirical estimation of variance by using the R packages DESeq2 [86] and fdrtool [87]. The genes were filtered if their read counts were less than three in every biological replicate. The genes were called significant if their adjusted p values by the false discovery rate (FDR) method were smaller than 0.05.
We performed gene set enrichment analysis (GSEA) to evaluate the enrichment of the gene sets available from MSigDB [88] after mapping genes to gene sets by gene symbols. The statistical significance of a gene set was calculated with the test statistics of individual genes computed by DESeq2. If the FDR-adjusted p value is less than 0.1, the term was called significant. We performed the GSEA analysis using the R package, CAMERA [89].
Allele-specific gene expression analysis
We performed allele-specific expression analysis as previously described [90]. For mouse testis samples, we created a C57BL/6J, Cast/EiJ diploid genome by incorporating single nucleotide polymorphisms and indels (obtained from the Mouse Genome Project: ftp://ftp-mouse.sanger.ac.uk/REL-1303-SNPs_Indels-GRCm38) from both strains into the M. musculus GRCm38 reference genome sequence. We created a transcriptome annotation set as follows. The gencode.vM2.annotation GTF file was downloaded, and Mt_rRNA, Mt_tRNA, miRNA, rRNA, snRNA, snoRNA, Mt_tRNA_pseudogene, tRNA_pseudogene, snoRNA_pseudogene, snRNA_pseudogene, scRNA_pseudogene, rRNA_pseudogene, and miRNA_pseudogene were removed (not enriched in our RNA-seq libraries). To create an extensive set of transcripts, we added to the gencode.vM2.annotation all transcripts from the UCSC knownGene mm10 annotation file, which are not represented in the gencode.vM2.annotation set. We also added all functional RNAs from the Functional RNA database (fRNAdb) [91], which did not intersect with any of the previously incorporated transcripts. From this, we then used the UCSC liftOver utility to generate a C57BL/6J, Cast/EiJ diploid transcriptome set.
Each RNA-seq library was first subjected to quality and adapter trimming using the Trim Galore utility (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) with stringency level 3. We then mapped each of the C57BL/6J::Cast/EiJ hybrid RNA-seq libraries to the C57BL/6J and Cast/EiJ diploid genome and transcriptome splice junctions using STAR RNA-seq aligner [83], allowing a maximum of 3 mismatches. The data were mapped twice, where after the first mapping step, we incorporated valid splice junctions that were reported by STAR to exist in the RNA-seq data. We then transformed the genomic alignments to transcriptomic alignments. Following that, we estimated the expression levels with their respective uncertainties for each transcript in our C57BL/6J and Cast/EiJ diploid transcriptome using MMSEQ [92]. The posterior FPKM samples were transformed into TPM units with a minimum expression TPM cutoff set to 0.01. In any RNA-seq sample, any transcript for which its MMSEQ posterior median TPM was lower than 0.01 was set to 0.01 (used as the minimal measurable expression level).
We adopted the approach of Turro et al. for combining lowly identifiable transcripts based on the posterior correlation of their expression-level estimates, tailored for a diploid transcriptome case [93]. In this approach, for any given RNA-seq sample, we compute the Pearson correlation coefficient of the posterior TPM samples of any pair of transcripts from the same locus and the same allele. Subsequently, if the mean Pearson correlation coefficient across all RNA-seq samples for a pair of transcripts in both alleles is lower than a defined cutoff (which we empirically set to − 0.25), each of these pairs is combined into a single transcript. This process continues iteratively until no pair of transcripts (or pairs of already combined transcripts) can be further combined. This consistency between the alleles in the combining process ensures that the resulting combined transcripts are identical for the two alleles and can therefore be tested for allelically biased expression.
Amplification of full-length Tug1
We amplified the full-length Tug1 isoform lacking the 5′ region (Ensembl ID: ENSMUST00000153313.2) from Riken cDNA clone E330021M17 (Source Bioscience) using specific primers containing MluI and EcoRV restriction sites (see the “Sequences and primers” section). After gel purification, the amplicon was sub-cloned, using the MluI and EcoRV restriction sites, into a modified Tet-On pTRE2pur vector (Clontech, 631013) in which the bGlobin-intron was removed. We verified the absence of mutations from the cloned Tug1 cDNA by sequencing using primers listed below. The plasmid was used also for sub-cloning Tug1 into pcDNA3.1(+) (see below).
RT-PCR
The testes were collected from wild-type and Tug1−/− mice (112 days old) and were homogenized in TRIzol (Invitrogen, 15596026) with a gentle MACS Dissociator (Miltenyi Biotec, 130-093-235). RNA was isolated by column purification (Qiagen, 74104) using a QiaCube (Qiagen). RNA was assessed and quantified on a Bioanalyzer (Agilent). cDNA was synthesized using 500 ng of total RNA as input with SuperScript IV VILO Master Mix with and without RT (Invitrogen, 11756050). PCR was performed on the synthesized cDNA (RT and no RT samples) using MyTaq Red Mix (Bioline, BIO-25043) with the cycling parameters: 95 °C for 1 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 10 s. PCR products were run a 1% agarose gel and verified by Sanger sequencing (GeneWiz). The following are the primers used for RT PCR: 7SK F1: GACATCTGTCACCCCATTGA and R1: TCCTCTATTCGGGGAAGGTC; Tug1 F1: CGGAGGAGCCATCTTGTCTTGTC and R1: GCTTCCAATTCCATACACACACTG; Tug1 F2: CTCTGGAGGTGGACGTTTTGT and R2: GTGAGTCGTGTCTCTCTTTTCTC.
ORF search
We analyzed human and mouse Tug1 cDNA sequences with CLC Genomics Workbench (Qiagen) for open reading frames (ORFs), allowing both canonical and non-canonical start codons (AUG, CUG, and UUG). After, sequences with annotated ORFs were aligned using MUSCLE alignment. All further sequence and amino acid alignments were performed with CLC Genomics Workbench.
Generation of human and mouse TUG1-BOAT overexpression constructs
We generated a synthesized construct for human Tug1 ORF1 that contained an in-frame 3xFLAG epitope tag prior to the stop codon, with and without the 5′ leader sequence (GeneWiz). We also synthesized a construct containing mouse ORF1 with an HA tag after the 3xFLAG before the stop codon, with and without the 5′ leader sequence (GeneWiz).
We amplified the Tug1 cDNA sequence with primers (see the “Sequences and primers” section) having KpnI and NotI restriction enzyme overhangs from the pTRE2-Tug1 vector plasmid using Q5 polymerase (Roche) and under the following conditions: 96 °C for 2 min, 35 cycles of (96 °C for 30 s, 65 °C for 30 s, 72 °C for 4 min), 72 °C for 4 min, and gel purified the amplicon. We digested the inserts and pcDNA3.1(+) plasmid with proper restriction enzymes according to the manufacturer’s instructions. After digestion, the plasmid was dephosphorylated using alkaline phosphatase. We then ligated the plasmid and inserts using T4 ligase (NEB) in a 1:3 ratio, respectively, followed by bacterial transformation, culture growth, and plasmid isolation (Qiagen Mini-Prep Kit).
Transfection of TUG1-BOAT constructs
We seeded 3T3 and HeLa cells in 10-cm plates containing poly-l-lysine-coated 18-mm glass coverslips. Next, we transfected the cells with 14 μg of plasmid (pcDNA3.1(+) containing each of the inserts) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) per manufacturer’s recommendations. Forty-eight hours post-transfection, cell pellets were harvested for protein extraction (see below) and coverslips were processed for RNA FISH and/or immunofluorescence (see below).
Protein extraction and western blot
We resuspended 3T3 and HeLa cell pellets in RIPA Lysis and Extraction Buffer 48 h post-transfection (Thermo Fisher Scientific). Total protein was quantified with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). We then separated a total of 20–25 μg of denatured protein on a 12.5% SDS polyacrylamide gel for 100 min at 120 V. We transferred proteins to an Immobilon-PSQ PVDF membrane (Sigma-Aldrich, ISEQ00010) at 400 mA for 75 min. After blocking in 5% dried milk in TBST, the membrane was incubated with properly diluted primary antibody (M2 Monoclonal ANTI-FLAG 1:1000, F1804, Sigma; Monoclonal GAPDH 1:5000, 2118S, CST) in 5% dried milk/TBST overnight at 4 °C. The next day, we washed the membrane three times for 5 min each in TBST (0.5% Tween-20). We then incubated the membrane with horse radish peroxidase-conjugated secondary antibody (anti-mouse 1:15,000, A9044, Sigma; anti-rabbit 1:10,000, 711035152, Jackson ImmunoResearch), diluted in 5% dried milk/TBST for 1 h at room temperature. Following three 5-min washes in TBST, SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermo Scientific, 34580) was added and chemiluminescence was detected using ImageQuant™ LAS 4000 imager. Original, uncropped images of western blots are provided in Additional file 16: Fig. S11.
TUG1-BOAT localization by immunofluorescence
We plated HeLa and 3T3 cells on poly-l-lysine-coated coverslips. Forty-eight hours post-transfection, we rinsed the coverslips twice with PBS and fixed the cells with 3.7% formaldehyde in PBS for 10 min at room temperature. After 2 washes with PBS, we permeabilized the cells with PBT (PBS, 0.1% Tween-20) for 15 min at room temperature. Next, we blocked the coverslips with 5% BSA in PBT for 1 h at room temperature and then incubated the coverslips with properly diluted primary antibody (mouse M2 monoclonal ANTI FLAG, 1:800, F1804, Sigma; rabbit polyclonal Tom20, 1:800, FL-145, Santa Cruz) in 5% BSA in PBT for 3 h at 37 °C in a humid chamber. Coverslips were washed three times for 5 min each with PBT and incubated with diluted secondary antibody (anti-mouse labeled with Alexa Fluor 488, 1:800, ab150113, Abcam; anti-rabbit labeled with Alexa Fluor 647, 1:800, 4414S, CST) in 5% BSA in PBT for 1 h at room temperature. Cells were then washed twice for 5 min with PBS, once for 20 min with PBS containing Hoechst DNA stain (1:1000, Thermo Fisher Scientific), rinsed in PBS, and then mounted on glass slides with ProLong Gold (Thermo Fisher Scientific).
Mitochondrial staining with MitoTracker® red chloromethyl-X-rosamine
We plated cells on poly-l-lysisne-coated coverslips and transfected as described in the previous sections. Forty-eight hours post-transfection, cells were incubated with 200 nM MitoTracker red chloromethyl-X-rosamine (Thermo Fischer Scientific, M7512) in 1 mL FBS-free growth media for 40 min. We then washed the cells twice with PBS, fixed with 3.7% formaldehyde for 10 min at room temperature, and processed for immunofluorescence and/or RNA FISH (as described previously).
Microscopy and image analysis
We acquired z-stacks (200 nm z-step) capturing the entire cell volume for single-molecule RNA FISH, single-molecule RNA FISH/CMXR staining, 3xFLAG tag immunofluorescence/CMXR staining, and/or Tom20 immunofluorescence with a GE wide-field DeltaVision Elite microscope with an Olympus UPlanSApo 100x/1.40-NA Oil Objective lens and a PCO Edge sCMOS camera using corresponding filters. 3D stacks were deconvolved using the built-in DeltaVision SoftWoRx Imaging software. Maximum intensity projections of each image were subjected for quantification using Fiji.
Fluorescence-activated cell sorting
Age- and sex-matched adult mice were used in all flow cytometry experiments. We obtained peripheral blood by cardiac puncture and collected blood into a 1.5-mL Eppendorf tube containing 4% citrate solution. Next, we added the blood-citrate mixture to 3 mL of 2% dextran/1× PBS solution and incubated for 30 min at 37 °C. The upper layer was transferred to a new 5-mL polystyrene FACS tube (Falcon, #352058) and centrifuged at 1200 rpm for 5 min at 4 °C. We then lysed red blood cells for 15 min at room temperature using BD Pharm Lyse (BD, 555899). Cells were washed twice with staining media (Hank’s Balanced Salt Solution (HBSS) containing 2% FBS and 2 mM EDTA). The following antibodies were added (1:100) to each sample and incubated for 30 min at room temperature: Alexa Fluor 700 anti-mouse CD8a (Biolegend, 100730), PE/Dazzle-594 anti-mouse CD4 (Biolegend, 100456), APC anti-mouse CD19 (Biolegend, 115512), Alexa Fluor 488 anti-mouse NK-1.1 (Biolegend, 108718), and PE anti-mouse CD3 (Biolegend, 100205), and Zombie Aqua Fixable Viability Kit (Biolegend, 423101) was used as a live-dead stain. We washed samples twice with staining media and sorted directly into TRIzol LS using a BD Aria FACS.
qRT-PCR
We isolated and quantified RNA from sorted blood populations as described in the RNA Isolation and RNA-Seq Library Preparation. One hundred nanograms of total RNA was used as input to generate cDNA using SuperScript IV VILO Master Mix (Invitrogen, 11756050), according to the manufacturer’s protocol. cDNA was diluted 1:3 with DNase- and RNase-free water, and 1 μL was used per each reaction. We performed qRT-PCR using FastStart Universal SYBR Green Master Mix with ROX (Sigma, 4913914001) on a ViiA 7 Real-Time PCR System (Thermo Fisher). Analysis was performed using the ∆∆Ct method [94]. Primers used in qRT-PCR experiments are listed in the “Sequences and primers” section.
GEO accession numbers
All primary RNA-seq data are available at the Gene Expression Omnibus (GSE124745 and GSE88819).