GSM5123415: RNA-seq of CRISPRoff with RAB11A targeting sgRNA, replicate 2 [RAB11A-GFP tagged line]; Homo sapiens; RNA-Seq
For RNA-seq, HEK293T cells that have maintained stable silencing of target genes were harvested 33 days (ITGB1 (CD29), CD81, and CD151) or 28 days (CLTA, HIST2H2BE, RAB11A) post CRISPRoff transfection. Cells were dislodged from plates with PBS, centrifuged at 500 × g for 5 min and washed again with PBS. Total RNA was extracted using Direct-zol RNA MiniPrep (Zymo R2051). For ChiP-seq, at 30 days post transfection, 10x106 cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with 1.25 M glycine. Crosslinked cells were washed twice with cold PBS containing 1% HaltTM protease inhibitors (Thermo Fisher Scientific) and the cell pellets were flash frozen at –80 °C until sample preparation. Cells were lysed in lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 1% Igepal, 1% protease inhibitors) for 10 min on ice. Nuclei were isolated after spinning the suspension at 2000 rpm at 4 °C for 5 min. Nuclei were lysed at 4 °C for 10 min in nuclei lysis buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS, 1% protease inhibitors). Chromatin shearing was performed at 4°C using a Diagenode Bioruptor® Pico sonication device in 1.5 ml Bioruptor® Pico Microtubes. The shearing program was optimized to obtain 200-700 bp fragments (30 seconds on, 30 seconds off for 10 cycles). The sonicated samples were centrifuged at 13,000 rpm at 4 °C for 10 min and the supernatant was collected and diluted 5-fold in IP dilution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Igepal, 0.25% deoxycholate, 1% protease inhibitors). A fraction of the input was saved and frozen (4% of total) prior to proceeding to immunoprecipitation. Immunoprecipitations were performed overnight at 4 °C using 5 µg of anti-H3K9me3 antibody (abcam ab8898). The washing steps were performed with PierceTM Protein A/G magnetic beads (Thermo Fisher Scientific) using the following protocol: once with IP wash buffer 1 (20 mM Tris pH 8, 2 mM EDTA, 50 mM KCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer 2 (10 mM Tris pH 8, 1 mM EDTA, 0.25 lithium chloride, 1% Igepal, 1% deoxycholate), and twice with TE buffer (10 mM Tris pH 8, 1 mM EDTA). Samples were eluted in elution buffer (50 mM sodium bicarbonate, 1% SDS) at 65 °C for 1 hour and reversed crosslinked initially in 300 mM NaCl and RNase A for 1 hour at 37 °C, followed by 63 °C overnight with Proteinase K (Thermo Fisher). The DNA samples were purified using a Zymo Clean & Concentrator kit. For RNA-seq, library preparations were carried out using TruSeq Stranded mRNA Library Preparation Kit (Illumina RS-111-2101), starting with 1000 ng total RNA. The ChIP-seq library were prepared using the NEBNext UltraTM II DNA Library Prep kit (NEB). The WGBS-seq libraries were prepared using the EZ DNA Lightning kit (Zymo, Cat. No. D5030) and the Swift Accel-NGS Methyl-Seq DNA Library Kit (Cat. No. 30024), starting from 1 ug DNA.