title: GSM9074416: hct116_wt_chip_input_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: 1% formaldehyde in DMEM was added to HCCT116 WT, DKO or TKO cells for 10 min, and quenched with 0.1 M glycine. Isolated nuclei were lysed and sonicated using a Bioruptor® Pico ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and pre-cleared with 10 μl packed sepharose beads for 2 h at 4°C. Pre-cleared chromatin was incubated with ChIP-grade antibodies against H3K9me3 or H3K27me3 overnight. 20 ng of D. melanogaster S2 chromatin and 0.5 μg of H2Av antibody were added as spike-in to each sample. Dynabeads™ Protein A (Invitrogen, 10002D) or G (Invitrogen, 10004D) were added. After 4 h at 4°C, beads were pelleted, washed, and bound chromatin eluted with 1% SDS, 100 mM NaHCO3. After digestion with Proteinase K (Thermo Scientific) at 50°C for 3 h and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform, ethanol precipitated and quantified by qPCR. To prepare samples for RNA-seq, 2 individual batches of cells were plated on the same day and further processed in parallel. Freshly harvested cells were lysed with 500 µL of QIAzol lysis reagent (QIAGEN #79306) and mixed by gentle pipetting. Subsequently, 100 µL of chloroform (Sigma) was added, samples were vortexed for 10 s and spun at 12'000 x g for 15 s at 4°C. The upper phase was transferred into a new tube, 600 μL of isopropanol (Sigma) added and samples incubated O/N at -20°C. After centrifugation at 16'000 x g for 10 min at 4°C, the RNA pellet was washed in 75% ethanol and resuspended in RNase-free water (Invitrogen). DNase digestion of RNA was performed using RNase-Free DNase Set (QIAGEN) following the manufacturer's instructions. RNA was then precipitated by adding sodium acetate (Sigma) to a final concentration of 0.3 M, and adding 3 volumes of 100% ethanol O/N at -20°C. RNA was recovered by centrifugation at 16'000 x g for 10 min and washed two times with 70% ethanol. RNA was dissolved in RNase-free water (Invitrogen). To prepare ATAC-seq samples, 3 individual batches of cells were plate on the same day in 6-wells (for si-11) or 10 cm plates (for KO cells). Cell batches were then further processed in parallel, during all subsequent steps. For harvest, cells were washed twice with 1x PBS and detached using 0.5 mM EDTA in 1x PBS. Cells were counted and 80'000 cells were pelleted. Pellets were resuspended in 200 µL of freezing medium (10% DMSO in FCS) and slowly frozen at -80°C. ATAC-seq was performed at QuickBiology (Monrovia, California). Here, cells were washed and lysed to generate a crude preparation of nuclei. This crude extract was then treated with Tn5 Transposase. Tn5 transposase tagmentation simultaneously fragments the genome and tags the resulting DNA with Illumina sequencing adapters. DNA fragments were PCR-amplified and subsequently purified using the MinElute PCR Purification Kit (Qiagen, Maryland, USA). For ChIP-seq analyses, the quantity of the isolated DNA was determined with Qubit® 4 Fluorometer (Life Technologies). Libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep for Illumina (New England Biolabs, E7645S and E7645L), following the manufacturer's protocol. Briefly, ChIP and input samples (15 ng) were end-repaired and polyadenylated. The adapters containing the index for multiplexing were then ligated. The quality and quantity of the enriched libraries were validated using 4200 TapeStation System (Agilent).