alias: GSM2046858
title: GSM2046858: ID44.H3K4me1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked then sonicated to generate chromatin fragment sizes between 250-500bp. For E2F1 and histone ChIP-seq, the chromatin was immunoprecipitated with appropriate amounts of antibody. For genomic input, the chromatin was not subjected to antibody pull-down. ChIP-enriched DNA was first quantified with the Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, P11496) and ChIP-seq libraries were prepared from 5 ng of ChIP DNA using the TruSeq ChIP Sample Preparation Kit Set A (Illumina, IP-202-1012) according to the manufacturer’s protocol with some modifications. Adapter-ligated DNA was amplified with 13 cycles using the Primer Cocktail and Master Mix provided in the kit. Amplified products of appropriate sizes were excised from agarose gel stained with SYBR® Gold Nucleic Acid Gel Stain (Invitrogen, S11494) and purified using the MinElute Gel Extraction Kit (Qiagen, 28604). The DNA libraries were then checked for quality and quantified using the Agilent 2100 Bioanalyzer and DNA 1000 Kit (Agilent Technologies, 5067-1504). The histone modification ChIP-seq libraries were sequenced by a local NGS service provider (Genome Institute of Singapore, Singapore) whereas the E2F1 ChIP-seq libraries were sent for sequencing to the Beijing Genomics Institute (BGI). All ChIP-seq libraries were sequenced as 36-bp single-end reads using the Illumina HiSeq 2000 according to standard manufacturer’s procedures. Total cellular RNA from UC-MSCs was extracted using TRIzol® Reagent (Invitrogen, 15 596-026) and chloroform before purifying with miRNeasy Mini Kit (Qiagen, 217004) according to the manufacturer’s instructions. RNA integrity was checked and concentration was determined using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Kit (Agilent Technologies, 5067-1511). 2 µg of total cellular RNA from the UC-MSCs was depleted of ribosomal RNA (rRNA) using the Ribo-ZeroTM Magnetic Gold Kit (Epicentre, MRZG126) as per manufacturer’s instructions and purified via ethanol precipitation. The removal of rRNA was confirmed using Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit (Agilent Technologies, 5067-1513). WT RNA-Seq libraries were then constructed using the TruSeq Stranded Total RNA LT Kit A (Illumina, RS-122-2201) according to manufacturer’s protocol. Adapter-ligated cDNA was amplified with 10 cycles using the PCR Primer Cocktail and PCR Master Mix provided in the kit. cDNA libraries were similarly checked for quality and quantified using the Agilent 2100 Bioanalyzer and DNA 1000 Kit as mentioned earlier before. The libraries were sent to Beijing Genomics Institute (China) for sequencing as 76-bp paired-end reads on the Illumina HiSeq 2000 according to standard manufacturer’s procedures.
identifiers.namespace: GEO
identifiers.id: GSM2046858