title: GSM3267051: ChIPseq_Input_TX1072_WT_12h_Dox_Rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were collected using Accutase (Thermo Fisher Scientific), washed twice in ice-cold PBS and counted. Typically, 3.5mln cells were used per immunoprecipitation (IP). A fraction of cells was always used for RNA/FISH verification of Xist induction. Cell pellet was resuspended in 90ul (per 10mln cells) of Lysis Buffer (50mM Tris-HCl, pH7.5; 150mM NaCl; 0.1% sodium deoxycholate; 1% Triton X-100; 5mM CaCl2; Protease Inhibitor Cocktail; 5mM sodium butyrate). After lysing cells on ice for 10min we added 62ul (per 10mln cells) of Lysis Buffer with MNase (500ul buffer + 0.5ul MNase). Chromatin was digested for exactly 10min at 37°C and reaction was stopped by the addition of 20mM EGTA. To remove uindigested debris the lysates were centrifuged at 13000rpm for 5min at 4°C. Supernatant was transferred to a fresh tube, an equal volume of STOP Buffer (50mM Tris-HCl, pH7.5; 150mM NaCl; 0.1% sodium deoxycholate; 1% Triton X-100; 30mM EGTA; 30mM EDTA; Protease Inhibitor Cocktail; 5mM sodium butyrate) was added, samples were stored on ice.  5ul of lysate was digested in 45ul of ProtK Digestion Buffer (20mM HEPES; 1mM EDTA; 0.5% SDS) for 30min at 56°C. 50ul of AMPure XP beads were added to the digested lysate together with 60ul of 20% PEG8000 1.25M NaCl. After mixing the samples were incubated for 15min at RT. Beads were separated on a magnet and washed twice with 80% Ethanol for 30sec. DNA was eluted in 12ul of Low-EDTA TE and DNA concentration was measured using Qubit DNA High-Sensitivity kit. These measurements were used to normalise lysate concentration between samples. DNA isolated in this step was used as the input sample. The volume of each undigested lysate was adjusted for equal concentration and to obtain 1mL per IP using a 1:1 mix of Lysis Buffer and STOP Buffer.  We have washed twice anti-mouse Dynabeads (50ul/IP) and Protein-A Dynabeads (10ul/IP) in Blocking Buffer (0.5% BSA; 0.5% Tween in PBS). Beads were then resuspended in Blocking buffer and coated with antibodies for 4hrs at 4°C (anti-mouse Dynabeads: H3K9ac[1ug/IP], H3K4me3[2.5ug/IP], H3K27ac[1ug/IP]; Protein-A dynabeads: H3K4me1[0.4ug/IP], H3K27me3[1ug/IP], H2AK119Ub[0.4ug/IP], H4ac[2ug/IP]). Once coated beads were magnet-separated and resuspended in 1mL of concentration-adjusted lysate. Samples were left rotating overnight at 4°C.  Following day beads were magnet-separated and washed quickly with ice-cold washing buffers. anti-H3K4me3 IP was washed 8 times with Low Salt Buffer (0.1% SDS; 1% TritonX-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; 150 mM NaCl; 0.1% sodium deoxycholate). All remaining IPs were washed 4-times with Low Salt Buffer, 2-times with High Salt Buffer (0.1% SDS; 1% TritonX-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; 360 mM NaCl; 0.1% sodium deoxycholate) and 2-times with LiCl buffer (0.25 M LiCl; 1% NP40;1.1% sodium deoxycholate; 1 mM EDTA; 10 mM Tris-HCl pH 8.1). Prior to elution all samples were rinsed once in TE. ChIP-DNA was eluted in ProtK-Digestion buffer for 15min at 56 °C. Beads were separated and the supernatant was further digested for another 2hrs at 56 °C. DNA was isolated using AMPure XP beads as described for the input sample.  For each nChIPseq, 0.5ul of each sample was used for qPCR validation of enrichment at control regions. 0.5ul of input samples were also used to verify the digestion efficiency using D1000 tapestation. Library preparation was done using Ovation® Ultralow Library System V2 following suppliers protocol. Amplified libraries were size-selected for dinucleotide fraction (350-600bp fragments) using agarose gel-separation and MinElute Gel Extraction Kit (Qiagen). Sample quality was inspected using D1000 tapestation. Samples were sequenced with Illumina HiSeq 2500 using PE100 mode.