GSM5176724: 2937_hoxb13-1; Homo sapiens; ChIP-Seq
Chromatin immunoprecipitation was performed using the ChIP-IT Express or ChIP-IT High Sensitivity kit (Active Motif) in accordance to the manufacturer's protocol. For prostate tissues, fresh normal or tumor specimens were cut into small pieces on dry ice and immediately crosslinked with 1% paraformaldehyde for 10 minutes. For cell lines, cells were grown in T-150 flasks and crosslinked with 1% paraformaldehyde at 70–80% confluency. Frozen cells or tissues were lysed using a Dounce homogenizer, and their chromatin was sheared by sonication to 1-kb fragments. Per IP reaction, up to 25 μg of sheared chromatin was incubated with 4 μg of antibodies and 25 μl of protein G magnetic beads at 4°C overnight. The captured antibody-bound protein/DNA complexes were extensively washed, and chromatin was eluted, reverse-crosslinked, and digested with proteinase K. The collected DNA was purified using a PCR purification kit (Qiagen) followed by qPCR analysis or high-throughput sequencing. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 3000 following the manufacturer's protocols. During library preparation a unique sequence tag called an index was added onto individual samples. This allows multiplexing all the samples together into a sequencing lane. Samples are run either as a single lane or multiple lanes of either type depending on the number of reads per sample required for analysis. To maximize for HOXB13 Transcription Factor binding sites in the ChIP sequencing we performed HiSeq3000 which obtains ~350M reads/lane at 1x50 read length. The DNA sequencing data was processed through a new pipeline that was generated and used for the ENCODE project.
GEO
GSM5176724