GSM6821283: Human11_RNAseq_ZIKVpos_24h; Homo sapiens; RNA-Seq
For RNA-seq, after virus infection of hDCs for 24 hours, cells were collected and stained with Zombie Violet™ Fixable Viability stain (BioLegend) for 20 min at 4˚C. After washing, cells were fixed and permeabilized with 4% paraformadehyde (PFA), 0.1% saponin in the presence of RNasin Ribonuclease inhibitor (400 U/mL). After centrifugation, samples were resuspended in wash buffer (0.2% BSA, 0.1% saponin and 400 U/mL of RNasin Ribonuclease inhibitor). Sample were blocked with human Fc blocker diluted in staining buffer (1% Bovine serum albumin (BSA), 0.1% saponin, 1600 U/mL RNasin Ribonuclease inhibitor) for 5 min, 4˚C. Then, 4G2-AF647 antibody was added to samples and incubated for 30 min, 4˚C. Samples were centrifuged at 1000xg for 3 min, 4˚C. Cells were washed using wash buffer and centrifuged at 1000xg for 3 min, 4˚C. Samples were resuspended in sort buffer containing 0.5% BSA and 1600 U/mL of RNase inhibitor in 1XPBS and sorted into ZIKV+ or DENV+ and ZIKV− or DENV- cells on a FACS cell sorter. After sorting, cells were pelleted at 4˚C, supernatant discarded and total RNA isolated using Ambion's RecoverAll Total Nucleic Acid Isolation Kit [AM1975], starting at the protease digestion step. All steps were performed per manufacturer recommendations with the following modification. Cells were incubated in digestion buffer for 3 hours at 50˚C supplemented with RNasin Plus RNase Inhibitor. RNA was treated with in column DNase per manufacturer instructions, eluted and RNA quality determined by BioAnalyzer using the Eukaryote Total RNA Pico Chip. Samples with RIN values greater than 8.0 were used for library preparation. For chromatin immunoprecipitation-sequencing (ChIP-seq) experiments, moDCs were cross-linked with 4 mM disuccinimidyl glutarate (DSG) in PBS for 30 minutes at RT followed by 1% formaldehyde for 15 minutes at RT and quenched with 0.125 M glycine. Preparation of moDCs for FACS was performed as described above for RNA-seq except that 1x cOmplete protease inhibitors (Roche) were included in all buffers instead of RNasin Plus RNase Inhibitor. After separation by FACS, cells were washed, separated into 500,000 cell aliquots, pelleted, snap-frozen and stored at -80°C. For RNA-seq, RNA libraries were generated using the TruSeq stranded total RNA-seq [Illumina] kit per manufacturer instructions and sequenced on an Illumina Hi-Seq 2000, NextSeq 500, or NovaSeq per manufacturer instructions. For ChIP-seq, samples of 500,000 cells were resuspended on ice in 500 µl RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5 mM DTT, 1x protease inhibitor cocktail). All subsequent steps were performed at 4°C. Chromatin was sheared by sonication in epitubes using 14 cycles of 10 s at ~13 W output and 30 s pause with a Sonic Dismembrator 60 (Fisher Scientific, Hampton, NH). Samples were recovered and spun at 21,000g for 5 min and the pellet was discarded. An aliquot of 2% of the sample volume was reserved as DNA input control and stored at −20°C. To prepare antibody-coupled beads for ChIP, protein A Dynabeads (20 μl per sample; Invitrogen cat. #10002D) were washed twice with 1 ml 0.5% BSA in TET (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.1% Tween 20), and resuspended in the same buffer. Anti-NFκB p65 antibody (C-20, Santa Cruz Biotechnology cat. # sc-372) was added to the beads and the sample was rotated for 1 h at room temperature. The supernatant was then removed and the Dynabeads were collected using a magnet, washed once with 0.1% BSA/TET, and resuspended in 10 µl RIPA buffer. For ChIP, 10 μl of prepared antibody-conjugated Dynabeads was added to each sample and rotated overnight at 4°C. The beads were then washed three times with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), three times with 10 mM Tris-HCl, pH 7.4, 250 mM LiCl, 1 mM EDTA, 1% Triton X-100, 0.7% sodium deoxycholate, and twice with TET/0.2% (as above except 0.2% Tween 20). Dynabeads were resuspended in 25 μl TT (10 mM Tris-HCl, pH 8, 0.05% Tween 20). Input samples were resuspended in 25 μl TT and libraries were generated in parallel with ChIP samples. Library NEBNext End Prep and Adaptor Ligation were performed using NEBNext Ultra II DNA Library Prep kit (New England BioLabs, cat. #E7645L) according to the manufacturer's instructions with barcoded adapters (NextFlex, Bio Scientific. Cat #514123). Libraries were incubated with proteinase K at 55°C for 1 h and then at 65°C overnight to reverse formaldehyde crosslinks. Libraries were PCR amplified for 14 cycles with NEBNext High Fidelity 2X PCR MasterMix (New England BioLabs, cat. #NEBM0541). Libraries were size selected for 200–400 bp fragments by gel extraction (10% TBE gels; Life Technologies) and were single-end sequenced on an Illumina NextSeq 500 (San Diego, CA). Samples were sequenced using the Illumina NextSeq 500 platform using 51 cycles (ChIP-seq). RNA-seq samples were single-end sequenced for 51 cycles on an Illumina NovaSeq 6000 or Hi-Seq 4000 (Illumina, San Diego, CA)