title: GSM3325203: Input HTT shRNA H9 hESCs (replicate 2); Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinked cells were quenched with 0,125M glycine for 10 min at room temperature and then scraped and transferred to a 15 ml conical tube on ice. Cells were centrifuged for 5 min at 4°C followed by two washing steps with 5 ml PBS 1x/ PMSF 1 mM. Cells were then resuspended sequentially in three different lysis buffers (lysis buffer 1: 50 mM Hepes, 140 mM NaCL, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100 and protease inhibitor (Roche); lysis buffer 2: 10 mM Tris, 200 mM NaCL, 1 mM EDTA, 0.5 mM EGTA; lysis buffer 3: 10 mM Tris, 100 mM NaCL, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-Lauroylsarcosine). Chromatin was then sonicated for 20 cycles (30 s on, 45 s off) using Bioruptor (Diagenode). After sonication, the material was centrifuged at 16000g during 3 min at 4°C, with the supernatant representing the sonicated chromatin. 75 μl was not subject to immunoprecipitation, thus representing total input control for the ChIP reactions. 750 μl were incubated with 10 μg of anti-H3K9me3 antibody (Abcam, #8898, reported suitable for ChIP) overnight at 4°C. On day 2, magnetic Dynabeads G (Thermofisher) at 10x volume of H3K9me3 antibody were aliquoted into a new microtube. Magnetic beads were washed five times with cold RIPA wash buffer (50 mM Hepes, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). Next, beads were washed once with 1 ml TE + 50 mM NaCl on ice and sample was centrifuged at 950g for 3 min at 4°C. After removing all liquid from beads, elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS) was added for 15 min at 65°C. Finally, beads were centrifuged for 1 min at 1600g and placed on magnetholder to settle and the supernatant was transferred into a new tube. To reverse crosslinking, 3x volume of the elution buffer was added to the input and left to incubate together with the ChIP sample at 65°C overnight. On day 3, 1x volume of TE buffer and 0.2 mg ml-1 RNase were added and incubated for 1 h at 37°C. Afterwards, 0.2 mg ml-1 Proteinase K was added and incubated for 2 h at 55°C to digest proteins. Next, DNA was phenol-chloroform extracted at room temperature with 1x volume of 25:24:1 phenol-chloroform-isoamyl alcohol and centrifuged for 5 min to separate layers, followed by the addition of 1x volume chloroform. The DNA was then transferred to a new tube for precipitation with 1/10 of NaOAc 3 M, 1 μl 20 mg ml-1 glycogen, 2x volume of ice cold ethanol during 30 min at -80°C. After a centrifugation during 30 min at 4°C, the supernatant was removed and we added 0.5 ml ice cold 70% ethanol followed by 5 min centrifugation. After removing the ethanol, the pellet was air-dried at room temperature and resuspended in 40 μl dH2O. Illumina ChIP Seq library Protocol
attributes.tag: GEO Accession
attributes.value: GSM3325203