title: GSM3080987: 2iFCS-input-spikeIn-13622; Mus musculus; ChIP-Seq
library_construction_protocol: For RNA-Seq, total RNA was isolated using a RNeasy mini kit (Invitrogen) according to manufacturer's instructions with on-column DNAseI treatment. Ribosomal RNA was depleted using Ribo-Zero Gold rRNGA Removal Kit (Illumina) according to manufacturer protocol starting from 5µg RNA. The resulting 180uL of rRNA depleted RNA was purified by mixing 18uL 3 M Sodium Acetate and 2 uL of 20 mg/mL glycogen, then adding 3x volumes of ice-cold 100% ethanol and incubation at -20°C for 1 hour. Samples were centrifuged at 10.000g for 30 minutes. After removal of supernatant, RNA pellet was washed in 500uL ice-cold 70% ethanol and centrifuged at 10.000g. Supernatant was removed and RNA was eluted in 40uL nuclease free H2O. rRNA depleted RNA was fragmented to ~200bp size using 10uL of a 5x 200mM Tris-acetate, 500 mM Potassium Acetate, 150 mM Magnesium Acetate, pH 8.2 solution at 95°C for 200 seconds, then incubated on ice for 10 minutes before repeating the ethanol purification procedure. First strand cDNA synthesis was performed using SuperScript III RT enzyme (Life Technologies), purified using Qiaquick MinElute Column (Qiagen). For ChIP-Seq, chromatin extracts were prepared by on-plate cell crosslinking in 1% PFA for 8 minutes. Crosslinking was quenched using 125 mM freshly dissolved Glycine final concentration. Fixed cells were washed in PBS twice, then collected by scraping. Pellets were lysed and sonicated in 50 mM Tris pH 8.0, 1% SDS and fresh protease inhibitor cocktail (Roche) at a density of 15 million cells per mL. The cells were sonicated in a Diagenode Bioruptor Pico for 8-10 30-second cycles. Proper sonication was verified using agarose gel size checks after decrosslinking and the eluted DNA was quantified using Qubit HS. 25ug DNA equivalent mouse chromatin was ChIPped using 4ug H3K27me3 (Millipore 04-779) or 2ug H3K36me3 (Diagenode pAb-192-050) antibody. Spike-in normalization was performed by adding 50ng Drosophila Melanogaster material (Active Motif 53083) and 2ug Drosophila-specific H2Av antibody (Active Motif 61686) to the mouse chromatin. ChIPs were diluted 9-fold using IP buffer (1% Triton X100, 1.2mM EDTA, 16.7mM Tris pH8.0, 167mM NaCl) and incubated overnight at 4°C while rotating. Subsequently, a mixture of 10uL Protein A and 10uL protein G (thermo) was blocked twice in IP buffer with 0.15% SDS and added to the ChIP. ChIPs with beads were left to incubate for 1 hour and precipitated using a magnetic rack. Chromatin was washed once (2mM EDTA, 20mM Tris pH8.0, 1% Triton, 0.1% SDS, 150mM NaCl), twice (2mM EDTA, 20mM Tris pH8.0, 1% Triton, 0.1% SDS, 500mM NaCl) and twice (1mM EDTA, 10 mM Tris pH8,0). Specific ChIPped DNA was eluted using 200mM NaCl, 1%SDS, 20mM Tris pH8.0 by shaking at 65°C for one hour, addition of and 1:100 10mg/mL Proteinase K and shaking at 55°C for one hour, then shaking at 65°C for at least four hours. The DNA was purified using Qiagen Minelute according to manufacturer instructions. For Bisulfite-seq, genomic DNA was extracted using the Wizard genomic DNA isolation kit (Promega). 200ng of purified genomic DNA was digested with Proteinase K before proceeding with sample processing for WGBS. For RNA-Seq, second strand synthesis was performed using incorporation of dUNTP's. Libraries were generated using Kapa Hyper Prep kit (KAPA Biosystems) according to manufacturer's protocol with modification of dUNTP cleavage before amplification. Libraries were size selected using Agencourt Ampure XP beads (Beckman Coulter). For ChIP-Seq, libraries were generated using Kapa Hyper Prep on 5ng DNA according to manufacturer instructions and size selected using Ampure XP beads. DNA was quantified using Qubit HS and DNA fragment sizes checked using Agilent Bioanalyzer HS. For WGBS, sample prep, paired-end sequencing and further analysis of Bisulfite-seq/WGBS was performed as in (Farlik et al., 2015).
attributes.tag: GEO Accession
attributes.value: GSM3080987