HES5::eGFP BAC transgenic human ES cells (H9; WA-09; Wicell) expressing GFP under the HES5 promoter
chip-antibody
none (Input)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP_Seq:To extract DNA and create the Illumina library we used AMPure XP beads (Agencourt) Solid-phase reversible immobilization (SPRI). SPRI beads were added to the samples, mixed 15 times, incubated at room temperature for 2 minutes. Supernatant was extracted from the beads from the beads 4 minutes on a magnet. We used 70% ethanol to wash the beads and then dried for 4 minutes. 40 ul EB buffer (10 mM Tris-HCl pH 8.0) was used to elute the DNA. ChIP_Seq:The following steps of Illumina library construction (end-repair, addition of A-base, ligation of barcoded adaptors and PCR enrichment) we used a general SPRI cleanup procedure: addition of PEG buffer (20% PEG and 2.5 M NaCl), and extracted and washed as above. The enzymatic reactions were carried as follows: 1. DNA end-repair: T4 PNK and T4 polymerase (New England Biolabs) incubated 12C for 15 min, 25C for 15 min; 2. A-base addition: Klenow (3’->5’ exonuclease; New England Biolabs) incubated at 37C for 30 min. 3. Adaptor ligation: DNA ligase (New England Biolabs) and indexed oligo adaptors and incubated 25C for 15 min, followed by 0.7X SPRI/reaction to remove non-ligated adaptors. 4. PCR enrichment: PCR mastermix (primer set, dNTP mix, Pfu Ultra Buffer (Agilent), Pfu Ultra-II Fusion (Agilent), water), for 20 cycles. The PCR amplified libraries we cleaned up using 0.7X SPRI/reaction (size selection mode) to remove excessive primers. Roughly 5 picomoles of DNA library was then applied to each lane of the flow cell and sequenced on Illumina HiSeq 2000 sequencers according to standard Illumina protocols.