title: GSM4119259: plc-WT atac-seq; Homo sapiens; ATAC-seq
library_construction_protocol: RNA-Seq:Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Double-stranded complementary DNAs were synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (Invitrogen). The cDNAs were then fragmented. ATAC-Seq: 50,000 cells for each sample were pelleted by centrifugation and lysed in lysis buffer containing 0.1% NP40, 0.1% Tween 20 and 0.01% Digitonin to obtain nucleic. The nucleic pellets were treated with TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) according to manufacturer's protocols. Immediately following transposition, DNA fragments were purified with MinElute PCR Purification Kit (QIAGEN) and PCR amplified with primers including barcode for a total of 10-12 cycles. The resulting libraries were purified and assessed with Qubit 3 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent). CHIP-Seq: cells were harvested and fixed. Chromatin was isolated by adding Lysis Buffer (Active Motif), followed by homogenization with Dounce homogenizer. Soluble chromatin was quantified by Nanodrop 2000 (ThermoFisher Scientific). ChIP was performed using Dynabeads Protein G (ThermoFisher Scientific) and 4 μL antibody against TCF4/TCF7L2 (Catalog number 2569, Cell Signaling Technology) with 20-50 μg soluble chromatins. Rabbit IgG (Catalog number ab172730, Abcam) was used for negative control. The beads-antibody-chromatin complexes were washed using ChIP-IT Express Enzymatic (Active Motif) according to manufacturer's protocols. The chromatin was reverse cross linked overnight at 65°C, treated with RNase A and proteinase K, respectively. The DNA was cleaned up with QIAGEN MinElute PCR Purification Kit (QIAGEN). RNA-Seq:using the Illumina paired-end RNA-seq approach according to the standard protocol. ATAC-Seq:The libraries were sequenced on Illumina Hiseq XTen instrument (Illumina).Briefly, 50,000 cells for each sample were pelleted by centrifugation and lysed in lysis buffer containing 0.1% NP40, 0.1% Tween 20 and 0.01% Digitonin to obtain nucleic. The nucleic pellets were treated with TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) according to manufacturer's protocols. Immediately following transposition, DNA fragments were purified with MinElute PCR Purification Kit (QIAGEN) and PCR amplified with primers including barcode for a total of 10-12 cycles. The resulting libraries were purified and assessed with Qubit 3 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent). CHIP-Seq: ChIP-seq libraries were prepared using KAPA Hyper Prep Kit and sequenced on Illumina Hiseq XTen instrument.Briefly, cells were harvested and fixed. Chromatin was isolated by adding Lysis Buffer (Active Motif), followed by homogenization with Dounce homogenizer. Soluble chromatin was quantified by Nanodrop 2000 (ThermoFisher Scientific). ChIP was performed using Dynabeads Protein G (ThermoFisher Scientific) and 4 μL antibody against TCF4/TCF7L2 (Catalog number 2569, Cell Signaling Technology) with 20-50 μg soluble chromatins. Rabbit IgG (Catalog number ab172730, Abcam) was used for negative control. The beads-antibody-chromatin complexes were washed using ChIP-IT Express Enzymatic (Active Motif) according to manufacturer's protocols. The chromatin was reverse c ross linked overnight at 65°C, treated with RNase A and proteinase K, respectively. The DNA was cleaned up with QIAGEN MinElute PCR Purification Kit (QIAGEN).
attributes.tag: GEO Accession
attributes.value: GSM4119259