title: GSM3937247: shMETTL3_WT_ssDRIP_rep2; Homo sapiens; OTHER
library_construction_protocol: gDNA was estracted and digested with cocktail of restrict enzymes MseI, DdeI, AluI, MboI, incubated at 37°C for 6 h. After purified by phenol/chloroform extraction, fragmented DNA was resuspended by TE, and quantified by Qubit 3.0 (Invitrogen). For each sample, 20 μg input DNA was immunoprecipitated overnight at 4°C in a shaker, with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 μg S9.6 antibody (ATCC, HB-8730). After adding 50 μl Dynabeads Protein G (Invitrogen, 10004D) and incubated for 4 h, the beads with antibody were washed by 1× DRIP binding buffer for 4 times at room temperature, each wash takes 10 min. Added 250 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to beads/antibody complexes, incubated for 40 min in an Eppendorf ThermoMixer at 55°C, 1,000 rpm. Purified by phenol/chloroform extraction, and moved supernatant to a new tube, followed by adding 1/10 volume 3 M NaAc, 1 μl GlycoBlue (Invitrogen) and 1 volume isopropanol, precipitated at -20°C for 3 h and then centrifuged. After washed by 70% ethanol and dried, the DRIPed DNA pellet was resuspended by 75 μl low EDTA TE buffer (10 mM Tris-HCl 8.0, 0.1 mM EDTA). NGS libraries were constructed using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences). DRIPed DNA sample was fragmented to an average size of 250 bp with an S220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). The sonicated DNA was denatured into ssDNA at 98°C for 2 minutes and placed on ice for another 2 min. Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences, 10096) was used. The truncated adapter 1 was ligated to 3' end of ssDNA first, followed by extension. The truncated adapter 2 was ligated to 5' end. After 12 cycles PCR reaction, libraries were purified by VAHTS DNA Clean Beads (Vazyme, N411). single strand DNA library
attributes.tag: GEO Accession
attributes.value: GSM3937247