title: GSM3593917: Satb1_ChIP_WT; Mus musculus; ChIP-Seq
library_construction_protocol: 10^7 Cells were washed once with DPBS and then fixed in 5 mL DMEM medium with 1% formaldehyde for 10 min at RT with rotation.  Fixation was terminated with addition of 100 mM glycine followed by 5 min rotation at RT. Cells were then scraped and spun down at 4 °C at 1000 rpm for 5 min, followed by two washes of ice-cold PBS. Cell pellets were resuspended in 1 mL Lysis Buffer 1 (50 mM Hepes-KOH, pH7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, and 0.25% Triton X-100) followed by rotation at 4 °C for 10 min. Cell pellets were spun down at 1400g for 5 min and resuspended in 1 mL Lysis Buffer 2 buffer (10 mM Tris-HCl, pH8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA). Following another 10 min incubation at 4 °C, cell pellets were again spun down at 1400g for 5 min and resuspended in 200 to 300 µL Lysis Buffer 3 (10 mM Tris-HCl, pH8.0, 100 mM NaCl, 1 mM EDTA,0.5 mM EGTA, 0.5% Na-Deoxycholate and 0.5% N-lauroylsarcosine).  Cells were then sonicated to about 500 bp DNA products using Covaris S200 machine with the following parameters: peak power=120, duty factor=2.0, cycle/burst=200, temperature=5°C and scale=10 min and a power output at 2.1.  The sonicated cell solution was then diluted with Lysis buffer 3 plus 1% Triton X-100, incubated for 15 min with rotation at 4 °C and spun down at 20,000g for 15 min. 10 µg anti-Satb1 antibodies (Abcam, Ab109122) were added into the supernatant with equivalent amount of Rabbit IgG antibody as controls.  After overnight incubation at 4 °C, antibodies were pulled down using anti-Rabbit dynabeads (10 µL per 1 µg antibody) after incubation with beads for 2 hr at 4 °C and dynabeads were extensively washed with RIPA buffer (50 mM Hepes-KOH, pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP40 and 0.7% Na-Deoxycholate) with rotation at 4°C for 3 times, 10 min each time.  After last RIPA wash, beads were washed once with 1×TE+50 mM NaCl buffer. DNA was then eluted in elution buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA and 1% SDS) twice at 65 °C for 20 min. Eluted DNA was then at 65 °C overnight for reverse cross-link. The solution was treated with RNase A1/T mix (thermo scientific) and incubated at 37 °C for 1 hr. This is followed by addition of 3 µL of proteinase K and incubation for 2 hr at 56 °C.  The solution was then phenol-chloroform extracted once followed by DNA extraction using MinElute PCR Purification Kit (28004, Qiagen).  DNA was eluted with EB buffer for downstream qPCR analysis or CHIP-seq library preparation. NEB NEBNext® Ultra™ DNA Library Prep Kit
attributes.tag: GEO Accession
attributes.value: GSM3593917