title: GSM2883847: ChIP-seq - Pan RNA Pol2 - Ter119+, APH treated Mouse spleen; Mus musculus; ChIP-Seq
library_construction_protocol: A single cell suspension was made by gently dissociating the spleen of a phenyhydrazine treated mouse and passing it through a 70um filter. Ter119 selection was performed by staining the cells with phycoerythrin conjugated anti-ter119 antibodies (BD biosciences). The cells were subsequently washed and then incubated with MACS anti-phycoerythrin beads. Cells were then separated using MACS LS columns (Miltenyi Biotec). 10 million cells aliquots were prepared and resuspended in 10ml of RPMI growth media with FBS (10%) at room temperature. Cells were fixed by the addition of formaldehyde to a final concentration of 1% and quenced using glycine (0.125M) for 5 minutes. Cells were pelleted (300g, 5 mins, 4°C), washed 1x in ice cold 1x PBS and then stored at -80°C until required. All stages were performed at 4°C on aliquots of 10 million cells, all buffers had protease inhibitors (Roche, 11 873 580 001) added to a final concentration of 1x; A cell pellet was resuspended in 200ul cell lysis buffer (PIPES pH 8.0 5mM,KCl 85mM, IGEPAL-CA 630 0.5%) and incubated on ice for 20 mins. Sample was spun at 900g for 10 mins and supernatant removed. Pellet was resuspended in nuclei lysis buffer (Tris-HCl (pH 7.5) 50mM, EDTA (pH 8.0) 10mM, SDS 1%) and incubated on ice for 5 mins. 30ul of buffer A (-SDS) (Tris-HCl pH 7.5 10mM, EDTA pH 8.0 1mM, EGTA pH 8.0 0.5mM, Triton X-100 1%, Na Deoxycholate 0.1%, NaCl 140mM) was used to the sample and mixed. The total sample (130ul) was transferrred to a clean Covaris microTUBE snap cap AFA fiber and sample was sonicated using a covaris S220 machine using an 8 minute program based on the manufatuters truChIP™ Chromatin Shearing guidelines. Sonicate was transferred to a clean 1.5ml safe lock eppendorf and spun @ 20,000g for 20mins, 4°C to pellet insoluable material. Supernatant was transferred to a clean tube and and diluted to 1.1ml using buffer A. 100ul of the diluted sonicated chromatin was taken and 50 buffer A added before freezing. The remaining chromatin was precleared for 2x 30mins on a rotating platform using 5ul of a 50:50 Protein A and Protein G dynabead slurry and 1ul of the sc899c antibody was separately conjugated to 100ul of dynabead slurry for 1hr. The precleared sonicate and antibody conjugated bead slurry were combined and left on the rotating platform for 24 hours @ 4°C. The immunoprecipitate was cleared using a magnet and supernatant discarded. Beads were washed using 500ul of the following buffers in the following order; 2x buffer A + SDS (Tris-HCl pH 7.5 10mM, EDTA pH 8.0 1mM, EGTA pH 8.0 0.5mM, Triton X-100 1%, Na Deoxycholate 0.1%, NaCl 140mM, SDS 0.1%), 2 x high salt RIPA buffer (Tris-HCl pH 7.5 10mM, EDTA pH 8.0 1mM, EGTA pH 8.0 0.5mM, Triton X-100 1%, Na Deoxycholate 0.1%, NaCl 500mM, SDS 0.1%),  1x LiCL RIPA buffer (Tris-HCl pH 7.5 10mM, EDTA pH 8.0 1mM, EGTA pH 8.0 0.5mM, Triton X-100 1%, Na Deoxycholate 0.1%, LiCl 250mM, SDS 0.1%) and 1x with 1x T.E (Tris-HCl pH 8.0 2mM, EDTA (pH 8.0) 0.5mM). One final wash in 1 x T.E was performed wihtout protease inhibitors. Beads were resuspended in 150ul elution buffer (Tris-HCl 20mM, EDTA (pH 8.0) 5mM, NaCl 50mM. Input sample was thawed and 20mg RNAse A added (Invitrogen) before mixing and incubating at 37°C on a shaking thermomixer 1000 RPM). 20mg Proteinase K was then added and temperature increased to 65°C and incubation continued overnight. Beads were magnetically separated and the supernantant subjected to phenol chloroform extraction followed by ethanol precipitation to yield 55ul of immunoprecipitate DNA for library prep. For input samples 5ng of DNA was used as an input for library prep. NEBNext Ultra DNA Library Prep Kit for Illumina was used to prepare libraries which were sequenced on a NextSeq550 (both procedures following manufacturers instructions)
attributes.tag: GEO Accession
attributes.value: GSM2883847