GSM3003918: CD4T_WT [ChIP-seq]; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) was performed as previously described (Goldberg et al., 2010; Lee et al., 2006). Briefly, 1x107 cells were cross-linked with 0.5% formaldehyde at room temperature for 10 min. Chromatin was sonicated to 300-500 bp in RIPA buffer with 0.3 M NaCl. 5-10 ug antibodies were pre-incubated with Dynabead Protein A/G (Invitrogen) for at least 8 hrs before incubating with sonicated chromatin overnight. After overnight incubation, beads were washed in modified RIPA wash buffer (100 mM LiCl) and 1x in TE. After overnight cross-link reversal at 65°C, RNase digestion and Proteinase K digestion, ChIP DNA and input DNA were purified using the Quiaquick PCR purification kit. For regular ChIP and for validation of ChIP-Seq, ChIP DNA was analyzed via qPCR using SYBR Green PCR Master Mix and the LightCycler 480. Primer sequences are available upon request. For ChIP-Seq, 30 µl of ChIP DNA were used to generate blunt-ended DNA using reagents supplied with the Epicenter DNA EndRepair kit (Epicenter Biotechnologies) according to the manufacturer's instructions. The end-repaired DNA was purified using the Quiaquick PCR purification kit. Using Klenow Fragment (3` to 5` exo-, NEB) “A” bases were added to the DNA. The DNA was purified using the MinElute kit. T4 DNA ligase (NEB) was used for ligation of Illumina/Solexa adapters to the DNA fragments. The adaptor-ligated DNA was purified with the MinElute kit. The DNA fragments were subjected to 18 cycles of PCR using the Illumina/Solexa primers 1.0 and 2.0 to generate the ChIP-Seq libraries. The ChIP-Seq libraries were purified with the MinElute kit.   Samples were sequenced on the Illumina Hiseq2000 platform for 50 cycles, and raw sequencing data were processed using the CASAVA_v1.8.2 software to generating fastq files. Sequencing reads were aligned to the mouse genome (mm9) using Bowtie v0.12.7 (Langmead et al., 2009). Reads were kept if they aligned with 2 errors or less and did not align to more than one location in the genome. A 25 bp density coverage map was created by extending each read for 100 bp to account for average library fragment length and mapping the number of reads per 25 bp bin using igvtools (Thorvaldsdottir et al., 2013). Values in each sample were normalized to fpkm values by calculating the fraction of mapped reads per bin in one million total reads.   For comparative analysis of promoter regions, the number of aligned reads in the area surrounding the transcriptional start site (+/- 3kb) of each gene was used.
GEO Accession
GSM3003918