title: GSM7901969: METTL3_KD_ChIRP_IP_rep2; Homo sapiens; OTHER
library_name: GSM7901969
library_construction_protocol: The cells were treated according to a previously published procedure. In brief, 1 × 107 cells were cross-linked with 1% formaldehyde for 15 min at room temperature, 0.125 M glycine was added, and the samples were incubated for 5 min at RT to stop the reaction. The cells were then snap-frozen using liquid nitrogen and stored at -80 °C. For each sample, the pellet was resuspended in 300 μL of nuclear lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) with the addition of 1.5 μL protease inhibitor and 1.5 μL PMSF. After centrifugation at 13,000 rpm for 30 seconds, the supernatant was removed. The pellet was resuspended in 200 μL of nuclear lysis buffer and the chromatin was sheared through 25 cycles (30s on/60s of) using a Bioruptor (Diagenode). Next, 1/30 volume (10 μL) of each sample was saved, 90 μL DNA reverse crosslinking buffer (20 mM Tris-Cl, 150 mM NaCl, 10 mM EDTA, 0.5% SDS) with 2 μL protease K was added, the samples were incubated overnight at 56 ℃, and DNA was extracted as the input DNA. The remaining sample was mixed with 1/4 volume of 5× rRIP Hybridization buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS, 1.5 M NaCl, 50% formamide). Next, biotinylated probes (5-30 pmol) were added to each sample, and the mixture was subjected to end-to-end rotation at 39 ℃ for 2-4 h. Subsequently, 20-80 μL well-washed streptavidin M280 beads were added to each sample (20 μL of beads for samples with 5 pmol probes; 40 μL of beads for samples with 20 pmol probes), followed by end-to-end rotation at 39 ℃ for 3 h. Following the pull-down, the beads were washed four times with 0.1× SSC washing buffer (0.1× SSC, 1% SDS) at 39 ℃, with each wash lasting 15-30 min. After the final wash, 100 μL freshly prepared 1× RNase H wash buffer (50 mM Tris-HCl, 75 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100) was added, and the mixture was shaken at 37 ℃ for 5 min. The samples were then transferred to a magnetic stand to remove the supernatant. The DNA was eluted with twice the bead volume of RNase H elution solution (NEB, M0297S) containing 0.2 U/μL RNase H, with shaking at 37 ℃ for 30 min. The elution tube was placed on the magnetic stand, and the elute was transferred into a new tube. The beads were resuspended in twice the bead volume of RNase H wash buffer with 1% SDS at RT and briefly vortexed for 5 s, and the supernatant was transferred and combined with the eluate mentioned above. To the elute, 5 M NaCl and 0.5 M EDTA were added to a final concentration of 100 mM NaCl and 10 mM EDTA, along with 1 μL of 20 mg/mL protease K. Reverse crosslinking was conducted at 56 ℃ overnight. The DNA was then purified using a PCR product purification kit (ZymoDNA Clean & Concentrator-500) following the manufacturer's instruction. Purified DNA samples were prepared for sequencing using the VAHTS Universal Pro DNA Library Prep Kit for Illumina. Library preparation was performed using the VAHTS Universal Pro DNA Library Prep Kit.