alias: GSM3680686
title: GSM3680686: ME3_control_1; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-Seq: Whole cell lysates were sonicated and protein-DNA complexes were isolated with antibodies. For RNA-Seq: Compound treated cells were collected and RNA was extracted using TRIzol. ChIP-sequencing for GAS41, c-Myc, H3K27cr and H3K27ac and RNA-sequencing in HCT116 cells with and without Dox treatment were performed on the Illumina HiSeq 2500v4 platform with 100bp read length under rapid run mode. Two to three biological replicates were prepared for each treatment condition. Specifically, for these genomic sequencing samples, ChIP-DNA was end repaired with T4 DNA polymerase and polynucleotide kinase. An A-base was added to the end-repaired DNA fragments. Solexa adaptors were ligated to the ChIP DNA fragments and 200-300bp size fractions were excised from 2% agarose gel stained with GelStar. Adaptor-modified fragments were enriched by 16 cycles of PCR amplification. The DNA library prep was validated in Bioanalyzer for quantity and size. Additionally, RNA-sequencing for HCT116 GAS41 wt and GAS41 KO cells, or GAS41 KO cells stably expressing GFP-Flag-GAS41 wt (wt rescue), or GFP-Flag-GAS41 Y74A/W93A (mutant rescue), was performed using an BGISEQ-500 platform at BGI. Three biological replicates were prepared for each treatment. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and single-end read of 50 bp were read through on the BGISEQ-500 platform for the following data analysis study. For this step, the BGISEQ-500 platform combines the DNA. nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method
identifiers.namespace: GEO
identifiers.id: GSM3680686