title: GSM1603220: S2P_Tat_ChIPSeq; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature in culture media, and the reaction was stopped by the addition of glycine (125 mM final).  Cells were washed twice with PBS and re-suspended in 1 ml of nuclei extraction buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitor (Roche)) at 2x107 cells/ml and incubated for 10 min at 4°C.  Cell nuclei were collected by centrifugation at 3000 rpm for 5 min at 4°C, and re-suspended in Szak's RIPA buffer (50 mM Tris-HCl pH 8.0, 1% NP-40, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 0.5 mM PMSF, protease inhibitor) at 5x107 nuclei/ml.  Nuclear pellets were sonicated using the Bioruptor water bath sonicator (Diagenode) using the high setting with 20 sec on 30 sec off for 45 min to generate 100-300 bp DNA fragments.  Chromatin DNA was quantified with NanoDrop 1000 (Agilent).  For the ChIP step, 1–3 mg of antibody was conjugated with 15 ml of protein G dynabeads (Invitrogen) and blocked with 0.16% bovine serum albumin for 16 hrs at 4°C.  Antibody-conjugated dynabeads were incubated with 30–50 μg of chromatin DNA for 2 hr at 4°C.  Then the beads were sequentially washed sequentially two times with the following buffers: low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 20 mM Tris, pH 8.0), and 1 mM Tris-EDTA pH 8.0.  Chromatin immunocomplexes were eluted by incubation for 10 min at 65°C with 1% SDS and 100 mM NaHCO3, and cross-linking was reversed by incubation in the solution adjusted to 200 mM NaCl and proteinase K (20 μg) for 1 hr at 65°C. ChIP DNA was submitted to McDermott Center Sequencing Core at UT Southwestern Medical Center for library preparation and high-throughput sequencing.  ChIP DNA was quantified on a Qubit® 2.0 Fluorometer (Invitrogen) and libraries from ~10 ng ChIP DNA were prepared using the SOLiDTM ChIP-Seq Kit with modifications for the 5500xl instrument (Applied Biosystems). Samples were end repaired, 3’-end adenylated and barcoded with multiplex adapters (Applied Biosystems).  After purification with Ampure XP beads (Beckton Coulter), samples were PCR amplified (~14 cycles), size selected with Ampure XP beads, and quantified on the Agilent 2100 Bioanalyzer.  Emulsion PCR was then performed and beads enriched on the EZ-Bead system.
attributes.tag: GEO Accession
attributes.value: GSM1603220