title: GSM1486230: Sak1_ChIPSeq; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: Variations introduced in the ChIP experimental method from Keogh et al. A) 1600 μl (60 ml initial culture) of prepared chromatin (TAP-tagged protein) and 20μl of beads (GE IgG Sepharose 6 Fast Flow) were used for IP. Pre-clearance of the beads with ascites fluid was not performed. IP was carried out for 2-3 hours at 4C. B) The following variations were introduced in the wash buffers used: Wash Buffer 1 – 1X FA-Lysis Buffer, 0.1% SDS, 1 M NaCl (Final Salt Concentration); Wash Buffer 2 – 1X FA-Lysis Buffer, 0.1% SDS, 500 mM NaCl (Final Salt Concentration). C) For each Wash Buffer, two rinses (invert 30 times) and one 4 minute wash on rotator was conducted. Beads were collected in between each step by centrifugation at 1000 rpm for 30 sec. While changing the Wash Buffer, beads were transferred to a fresh microcentrifuge tube (total 3 tube changes). D) Purified IP’ed DNA was re-suspended in 40 μl DNase free water. 10 μl of this IP’ed DNA was used for qPCR’s (Roche Lightcycler 480 SYBR Green I Master, Machine: Eppendorf Realplex2 Mastercycler ep Gradient S) and Illumina sequencing libraries were made from the remaining 30 μl. A) End Polish (final conc.): IP’ed DNA, 1X T4 DNA Ligase Buffer (NEB), 3U T4 DNA Polymerase (NEB), 10U T4 Polynucleotide Kinase (NEB), 0.4 mM dNTP’s. Reaction was kept at 20C for 30 minutes. It was subsequently cleaned up with QIAquick PCR Purification Kit and eluted in 20 μl water. B) A-tail addition (final conc.): Previously eluted DNA, 5U Klenow Fragment (3’-5’ exo-) (NEB), 33.33 μM dATP, 1X NE Buffer 2 (NEB). Total volume was 30 μl. The reaction was incubated at 37C for 30 minutes. The enzyme was inactivated by heating at 75C for 20 minutes and was ramped down (rate=10%) to 4C. C) Adaptor Ligation (final conc.): previous volume, 750U T4 DNA Ligase (Rapid) (Enzymatics), 1X T4 DNA Ligase Buffer, 37.5 nM Illumina adaptor (sequences and detailed method for adaptors, below) (Iossifov et al., 2012). The reaction was kept at 25C for 1 hr and inactivated at 65C for 10 minutes. The reaction was cleaned up twice using 0.8X volume of Agencourt Ampure beads (1st elution volume: 40 μl H2O, second elution volume: 15 μl H2O). D) PCR Amplification: eluted DNA, 1X Phusion Master Mix (NEB M0531), 2U Phusion Enzyme (NEB M0530), 200 μM dNTP’s, 1 μM each library amplification primers. The libraries were amplified between 18-21 cycles (annealing temp., 65C for 30 sec.; polymerization time, 30 sec.). E) The amplified products were purified using 0.8X volume of Agencourt Ampure beads. Eluted DNA was size-selected using 2% agarose gel electrophoresis followed by cutting out the 250-350 bp region. The DNA was purified from the gel using QIAquick Gel Extraction kit (Qiagen). Eluted DNA was quantitated on Qubit (Invitrogen) and size range was checked using Bioanalyzer 2100.
attributes.tag: GEO Accession
attributes.value: GSM1486230