GSM6254307: RNAseq.HBEC.C12.Rep2; Homo sapiens; RNA-Seq
Omni-ATAC protocol was used to probe DNA accessibility with slight modifications covered below 54. 100,000 cells per sample were trypsinized and washed with cold PBS to remove trypsin. Cell pellets were lysed in 50μl cold resuspension buffer (RSB) supplemented with fresh NP40 (final 0.1% v/v), Tween-20 (final 0.1% v/v), Digitonin (final 0.01% v/v) (RSB recipe: 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2). Lysis step was quenched with 1 mL of RSB supplemented with Tween-20 (final 0.1% v/v) and nuclei were pelleted at 500g for 10 min at 4°C after incubating on ice for 3 minutes. Nuclei were resuspended in 50μl transposition reaction mix containing 25 μl 2X Tagment DNA buffer (Illumina), 2.5μl Tn5 transposase, 16.5μl 1X PBS, 0.5μl 1% digitonin (final 0.01% v/v), 0.5μl 10% Tween-20 (final 0.1% v/v), and 5μl nuclease-free water. Transposition reaction was incubated at 37°C for 30 min with constant shaking (1000 rpm) on a thermomixer. Tagmented DNA was purified using the Qiagen MinElute Reaction Cleanup Kit. Standard ATAC-seq amplification protocol with 7 cycles of amplification was used to amplify tagmented libraries. Libraries were sequenced on Next-seq 500 (Illumina) using 37 bp pair-end sequencing. RNAs were extracted using Direct-zol RNA MiniPrep Kit and submitted to the Yale Center for Genome Analysis for library preparation. RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 instrument with the goal of at least 25 × 106 reads per sample. Reads were aligned to reference genome chlSab2, NCBI annotation release 100, using STAR aligner v2.7.3a(Dobin et al., 2013) with parameters --winAnchorMultimapNmax 200 --outFilterMultimapNmax 100 --quantMode GeneCounts. Differential expression was obtained using the R package DESeq2 v1.32(Dobin et al., 2013; Love et al., 2014). Bigwig files were generated using deeptools v3.1.3(Ramírez et al., 2016) with parameter --normalizeUsing RPKM CUT&Tag was carried out using a protocol developed by Epicypher in 8-strip PCR tubes with slight modifications as described below. Briefly, Concanavalin A (ConA) coated magnetic beads were activated with Bead Activation Buffer containing 20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2; beads were stored on ice until used. 300,000 cells/sample were trypsinized and pelleted by centrifugation at room temperature (600g for 3 min). Cells were lysed using cold Nuclear Extraction Buffer containing 20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol supplemented with fresh 0.5 mM Spermidine, and cOmplete, Mini, EDTA-free Protease Inhibitor (Roche) for 2 min. Nuclei were pelleted by centrifugation (600g for 3 min), resuspended in 100ul/sample Resuspension buffer (20 mM HEPES–KOH, pH 7.9, 150mM NaCl supplemented with fresh 0.5 mM Spermidine, and cOmplete, Mini, EDTA-free Protease Inhibitor) and incubated with activated ConA beads at room temperature for 15 min. The nuclei-ConA bead complex was resuspended in Antibody 150 Buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, supplemented with fresh 0.5 mM Spermidine, Protease Inhibitor (Roche), 0.01% Digitonin, and 0.5ug primary antibody/sample. Following overnight incubation at 4°C on a nutator, supernatant was discarded, and beads were washed once with Digitonin 150 Buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, supplemented with fresh 0.5 mM Spermidine, Protease Inhibitor (Roche), and 0.01% Digitonin. The ConA-nuclei complexes were then incubated with Digitonin 150 buffer supplemented with 0.5ug/sample Secondary antibody for 1hr at room temperature on a nutator. They were then washed with Digitonin 150 Buffer twice before resuspension in 50uL cold Digitonin300 Buffer containing 20 mM HEPES, pH 7.5, 300 mM NaCl, supplemented with fresh 0.5 mM Spermidine, protease inhibitor, and 0.01% Digitonin. 2.0 µL CUTANA pAG-Tn5 (Epicypher) was added to each sample and incubated on a nutator for 1hr at room temperature. Following incubation, beads were washed twice with cold Digitonin300 Buffer. Targeted chromatin tagmentation and library amplification were carried out according to Epicypher's protocol. RNA-seq libraries were prepared with Illumina's TruSeq standard mRNA Sample Prep Kit using standard protocols. ATAC-seq libraries were prepared using a standard protocol (Buenrostro et al., Current Protocols (2013)).