title: GSM4805407: WT_HCT116_R1; Homo sapiens; RNA-Seq
library_construction_protocol: For ChIPseq, cells were harvested and washed with ice-cold PBS for two times, and fixed with paraformaldehyde (1% final) for 10 min at RT. Then quench the paraformaldehyde with 2.5M (1/20) glycine and wash the cell pellets twice with PBS. Sonication: the cell pellet was resuspended with lysis buffer 1 (50 mM HEPES, pH=7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 X protease inhibitors) and incubated on nutator at 4 °C for 10 min. Spin down at 500 g for 5 min and discard supernatant. The pellet was washed with lysis buffer 2 (10 mM Tris-HCl, pH=8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 X protease inhibitors) and resuspended with lysis buffer 3 (10 mM Tris-HCl, pH=8.0, 1 mM EDTA, 0.1% SDS, 1 X protease inhibitors). Adjust the final volume to be 10 times of the pellet with lysis buffer 3. Sonication with 1 ml Covaris tubes at 10% duty cycle, 175 peak intensity power, 200 cycles per burst for 60-1200s. Sonicated DNA size was checked on a 2% agarose gel. We next added 10% of 10X ChIP Dilution Buffer (10% Triton x-100, 1 M NaCl, 1% Na-Deoxycholate, 5% N- Dauroylsarcosine, 5 mM EGTA) to the lysate and spun at 20,000 g for 15 min at 4 °C to pellet debris. Supernatant was collected and the concentration measured by nanodrop. A small aliquot was saved as input. Antibody was added (~ 10 ug per antibody) to each cell lysate and incubated at 4 °C on nutator overnight. The next morning, protein A/G agarose beads (Santa Cruz) were washed in RIPA buffer (50 mM HEPES, pH=7.5, 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate) before being incubated with chromatin/antibody solution at 4 °C on nutator for 4 hours. Samples were next spun down in 15 ml conical tubes at 200g for 5 min at 4 °C and supernatant was discarded. Beads were pelleted in 15 ml conical tubes at 200g for 5 min at 4 °C and washed in RIPA Buffer 4 times at 4 °C on nutator for 10 min each time. Beads were washed once with 1 ml of cold TE + 50 mM NaCl and spun at 1000 g for 3 min at 4 °C and remove any residual buffer. Beads were resuspended in 200μl Elution Buffer (50 mM Tris-HCl, pH=8.0, 10 mM EDTA, 1.0% SDS) and incubated in 65 °C water bath for 30 min, vortexing gently twice in between. Beads were spun down at 1000 g for 3 min at RT and supernatant was transferred to a new tube. To reverse crosslinks, a 50μl aliquot was diluted in 150 μl (3 volumes) Elution Buffer and incubated at 65 °C for 6-15 h. We added 4 μl of 20 mg/ml Protease K and incubated at 55 °C for 2 h to degrade protein. DNA was purified using Qiagen PCR Purification columns. For RNA-seq, polyA strategy, we use the NEBNext PolyA mRNA Magnetic Isolation Module along with the NEBNext Ultra II Directional RNA Library Prep Kit. ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit