title: GSM1290246: HPT.Blue.ChIPSeq.R1.ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Preparation of solubilized chromatin fractions from these cells (Fritsch et al. Mol. Cell 2010, PMID: 20129054) and tandem affinity purification (Lebreton et al. Science 2011, PMID: 21252314) were performed as previously described. Only the eluates from the first affinity column were used for DNA extraction. DNA was purified by two extractions with equal volumes of phenol:chloroform:isoamy lalcohol (25:24:1, pH=8), starting from 10 µL of the input samples diluted in 200 µL water, or 200 µL of eluates. Phenol was eliminated with one extraction with chloroform, then DNA was precipitated by addition of 900 µL of ethanol, 100 µL of ammonium acetate 7.5 M and 0.2 µL of glycogen (20 µg/µL) to each aqueous phase, incubation at -80°C for 30 min and centrifugation at 4°C, 20,000  ´ g for 15 min. The pellet was washed once in 100 µL of ethanol, then resuspended in 50 µL of pure water for inputs, 25 µL for eluates. 2 independent experiments were performed (R1 and R2) The library construction was performed by Fasteris according to the Illumina guidelines, with the following steps: 3’ A addition; Ligation of adapters; Gel purification to isolate fragments of specified inserts size; PCR amplification to generate the DNA Colonies Template Library, library purification; Library quantification and dilution at 10 nM. Library Quality Control, done by titration run on the HiSeq. Independent libraries were produced for R1 and R2. The DNA Colony Template library was sequenced as single reads of 50 bp. Sequencing was multiplexed at 3 libraries per channel. For the HPT-BAHD1 ChIP sample, the R2 library was sequenced twice (R2.a and R2.b).
attributes.tag: GEO Accession
attributes.value: GSM1290246