title: GSM9135568: SC shCTCF-2, replicate 1, ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: The experiments were performed following the method described in a previous study (Liu, Y., Shi, M., He, X., Cao, Y., Liu, P., Li, F., Zou, S., Wen, C., Zhan, Q., Xu, Z. et al. (2022). LncRNA-PACERR induces pro-tumour macrophages via interacting with miR-671-3p and m6A-reader IGF2BP2 in pancreatic ductal adenocarcinoma. J Hematol Oncol 15, 52. 10.1186/s13045-022-01272-w.). For MeRIP-seq, Total RNA (30 μg) was purified using DynabeadsTM Oligo(dT)25 (Thermo Fisher, USA) in two rounds to isolate poly(A) RNA. Subsequently, the purified poly(A) RNA was fragmented using the Magnesium RNA Fragmentation Module (NEB, USA, catalog number E6150S) at 86°C for 7 minutes. The fragmented RNA was then incubated with an m6A-specific antibody (Synaptic Systems, Germany, catalog number 202003) in IP buffer (50 mM Tris-HCl, 750 mM NaCl, and 0.5% Igepal CA-630) at 4°C for 2 hours. Next, the IP RNA was reverse transcribed into cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, USA, catalog number 1896649). PCR amplification was performed with the following conditions: initial denaturation at 95°C for 3 minutes, followed by 8 cycles of denaturation at 98°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The average insert size of the final cDNA library was 300 ± 50 bp. The cDNA library was subjected to paired-end sequencing (PE150) using the Illumina HiSeq X Ten platform (LC-Bio Technology Co., Ltd., Hangzhou, China). For ChIP-seq, the ChIP-seq library was prepared using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech Co., Ltd, ND607) according to the manufacturer's instructions. The generated DNA library was sequenced using the Illumina HiSeq X Ten sequencing system in paired-end mode. For RNA-seq, Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RNA content and purity of each sample were measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). The integrity of the RNA was evaluated using the Bioanalyzer 2100 (Agilent, CA, USA) to ensure the RNA Integrity Number (RIN) value greater than 7.0, and further validated by denaturing agarose gel electrophoresis. Two rounds of purification were performed using Dynabeads Oligo(dT)25-61005 (Thermo Fisher, CA, USA) to purify poly(A) RNA from 1 μg of total RNA. Subsequently, the poly(A) RNA was fragmented using the Magnesium RNA Fragmentation Module (NEB, USA, catalog number e6150) at 94°C for 5-7 minutes. The fragmented RNA was then reverse transcribed into cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, USA, catalog number 1896649). The fragmented RNA was used as a template for cDNA synthesis, which was subsequently converted into U-labeled second-strand DNA using E. coli DNA polymerase I (NEB, USA, catalog number m0209), RNase H (NEB, USA, catalog number m0297), and dUTP Solution (Thermo Fisher, USA, catalog number R0133). A base A was added to the end of each strand to prepare for ligation to the index adapters. Each adapter contained a T-overhang, which allowed the adapter to be ligated to the A-tailed fragment DNA. Single or dual index adapters were ligated to the fragments, followed by size selection using AMPureXP beads. After treatment with thermosensitive UDG enzyme (NEB, USA, catalog number m0280) to remove the U-labeled second-strand DNA, PCR amplification was performed. The amplification conditions included an initial denaturation step at 95°C for 3 minutes, followed by 8 cycles of denaturation at 98°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The average insert size of the final cDNA library was 300 ± 50 bp. Finally, according to the supplier's recommended protocol, paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000 platform.