title: GSM3307827: 34 hour IP #2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixed on plate with 1% PFA in PBS for 10 minutes at RT. Glycine was added to a final concentration of 125mM. Cells were washed 2 times with 1x PBS with protease inhibitor cocktail (PIC, Roche, 11836170001). They were lysed on the plate with cold Farnham lysis buffer to ~10x10^6 cells /mL (5mM PIPES pH 8.0, 0.5% NP-40, 85mM KCl,1mM EDTA, PIC) and incubated rotating for 15min at 4°C . Lysates were scraped off the plates, pelleted and resuspended in LB2 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM 128EDTA, 0.5 mM EGTA, PIC) and incubated rotating for 15 minutes at 4°C and then centrifuged. Pellets were resuspended to 5x10^7 cells/mL in LB3 (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium-deoxycholate, 0.5% sodium lauroyl sarcosinate, PIC) until suspension was homogenized. Samples were then sonicated using Covaris ultrasonicator model S220 for 15 minutes with the following settings: 140W peak power, 5% duty, 200 cycles per burst. TritonX-100 to a final concentration of 1% was added to the samples. Samples were clarified by centrifugation at 20,000 g for 10 minutes at 4°C. The supernatant is the soluble chromatin extract. The soluble fragmented chromatin from ~2.5x10^7 was used for each IP. For each IP 100ul Dynabeads (Thermofisher anti-rabbit M280, 11203D) were mixed with 10ul polyclonal rabbit-APOBEC2 antibodies (gift from Alin Vonica MD, PhD) incubating overnight (~16 hours). A magnetic stand was used to separate beads from the lysate and beads were washed one time each with for 5min in: low salt wash (0.1%SDS, 1%Triton X-100, 2mM EDTA, 20mM Tris pH8, 150mM NaCl, PIC), high salt wash (0.1%SDS, 1% Triton X- 100, 2mM EDTA, 20mM Tris pH8, 500mM NaCl, PIC), lithium chloride wash (150mM LiCl, 1% NP-40, 1% NaDOC, 1mM EDTA, 10mM TrispH8, PIC), TE wash (10mM Tris-HCl pH8, 1mM EDTA, 50mM NaCl, PIC). Beads were resuspended in 52 ul of elution buffer (50mM Tris-HCl pH8, 10mM EDTA, 1%SDS) and incubated at 30min at 65°C while shaking to prevent beads from settling. The eluate was transferred to a new tube, inputs of the same volume were incubated for 8 hours at 65°C to reverse the crosslink. The samples were treated with RNAse (Roche, Cat. No. 11 119 915 001) for 1 hour at 37°C, and with Proteinase K for 2 hours at 55°C. Fragmented DNA was purified 129 with Ampure beads (Agencourt AMPure XP beads A63881) as per the manufacter's instructions. ChIP-Seq libraries were prepared using NEBNext Ultra DNA Library Prep Kit as per manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3307827