GSM6261619: HEKcells_siFtreated_Input_rep1; Homo sapiens; OTHER
Between 7 and 9 x 106 cells were harvested at 72 hours post-siRNA treatment, and cell pellets frozen in liquid nitrogen. Pellets were later resuspended in 1.6 mL of TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA) that was added to include 0.6% sodium dodecyl sulfate (SDS) and 60 µg/mL Proteinase K. After Proteinase K digested lysates overnight at 37 ºC, DNA was extracted using the MaXtract High Density tubes (Qiagen, 129065). DNA was spooled by glass Pasteur pipette, transferred, then ethanol precipitated. DNA was digested overnight in TE buffer with 1x NEBuffer™ 2 (New England Biolabs Inc., #B6002), BSA (95 µg/mL), spermidine (1 mM), and 30 U each of restriction enzymes BsrGl, EcoRI, HindIII, SspI, and XbaI. Efficient restriction digest was confirmed by agarose (0.8% w/v) gel electrophoresis. Samples were sonicated for 30 cycles (30 sec on / 30 sec off). Negative control samples were digested by 20U RNaseH (Fisher Scientific, 50-811-717) per 10 µg DNA. After restriction enzyme and RNaseH digests, DNA was extracted by Phase Lock Gel™ (VWR, 10847-800) per manufacturer instructions. 50 µg of DNA was incubated overnight at 4 ºC with 20 µg S9.6 antibody and in 500 µL DRIP buffer (1x TE buffer, 10 mM Na phosphate pH 7, 140 mM NaCl, 0.05% Triton X-100). 5 µg of DNA was kept for input samples. Antibody precipitated with 100 µL Protein A/G beads (Millipore, IP05-1.5ML) was washed twice in 700 µL DRIP buffer. Hybrids were eluted by incubating beads with 300 µL DRIP Elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 0.5% SDS, 0.5 µg/µL Proteinase K) for 45 minutes at 55 ºC, then extracted by Phase Lock Gel™. Sample libraries were prepared and sequenced to by Novogene Corporation Inc. using a NovaSeq 6000 with 150 base paired-end reads (8 G raw data per sample).