title: GSM3132959: CV5 H3K27ac; Mus musculus; ChIP-Seq
library_construction_protocol: Organoids were harvested by using mechanical dissociation followed by multiple ice-cold PBS washes (400 × G, 4°C, 5 min). For RNA-seq, total RNA was extracted with Rneasy RNA extraction kit (Qiagen). For ATAC-seq, organoids were resuspended in Recovery Cell Culture Freezing Medium (Gibco) and snap frozen prior to the ATAC-seq procedure. Once thawed, they were washed with PBS and processed for ATAC-seq. For ChIPmentation, cell pellets were dissolved in PBS and crosslinked in solution by using formaldehyde (1% final volume, shaking 10 minutes at room temperature). Glycine (0.125 M) was added to quench the reaction. Cell pellets were lysed in a volume of 110 μL using lysis buffer (20mM Hepes pH 7.6, 1% SDS, 1x Protease Inhibitor Cocktails. Samples were sonicated using the Biorupter Pico (Diagenode) with 6 cycles (30 sec on / 30 sec off). Afterwards the samples were spun at 16.200×G for 5 min at room temperature and stored at -80°C prior to the ChIPmentation procedure For RNA-seq experiment,  RNA was purified and ERCC spike-in was added. rRNA was depleted using RiboZero kit. rRNA depleted samples were fragmented for 5 min at 95°C using 5× fragmentation buffer (200mM Tris-acetate, 500mM Potassium Acetate, 150mM Magnesium Acetate, pH8.2) followed by ethanol precipitation. First strand cDNA synthesis was performed using Superscript III (Invitrogen) with the addition of random hexamer primers and Actinomycin D (9ng/mL). Afterwards second strand cDNA synthesis (SuperScript, Invitrogen) was performed with the addition of dUNTPs and random hexamer primers. Samples were cleaned using Qiaquick MinElute Columns (Qiagen) after first and second strand cDNA synthesis. A maximum of 5ng cDNA was used for sample preparation using the KAPA Hyper Prep Kit (KAPA Biosystems). NextFlex DNA barcodes (Bioo Scientific) were used for adapter ligation. Treatment for 15 min at 37°C with USER enzyme was done prior to library amplification. Libraries were amplified using 10 amplification cycles. Post-amplification cleanup was done using Qiaquick MinElute Columns (Qiagen) followed by size selection of 300 base pairs using E-Gel SizeSelect Agarose Gels 2% (Invitrogen). For ChIPmentation the chromatin was incubated overnight at 4°C rotating in dilution buffer (1% Triton X-100, 1.2mM EDTA, 16.7mM Tris pH8, 167mM NaCl), 1x Protease Inhibitor Cocktail (Roche), and 1 μL of antibody (H3K4me3 / H3K27ac / H3K27me3, Diagenode). Per ChIP 10 μL protein A Dynabeads and 10 μL protein G Dynabeads were used. Beads were washed using dilution buffer (+0.15% SDS, +0.1% BSA) and incubated with the chromatin and antibody mix for 60 minutes at 4°C rotating. Afterwards the beads were washed at 4°C once with ChIP wash buffer 1 (2mM EDTA, 20mM Tris pH 8, 1% Triton X-100, 0.1% SDS, 150 mM NaCl), twice with ChIP wash buffer 2 (2mM EDTA, 20mM Tris pH8, 1% Triton X-100, 0.1% SDS, 500 mM NaCl), and twice with TE (1 mM EDTA, 10 mM Tris pH 8). Beads were resuspended in Tagment DNA buffer and 1 μL Tn5 enzyme (produced in-house) and incubated for 10 minutes at 37°C with 550 rpm shaking. Afterwards the beads were washed twice with WBI (20 mM Hepes, 150 mM NaCl, 0.1% SDS, 0.1% DOC, 1% Triton X-100, 1 mM EDTA, 0.5mM EGTA) and twice with WBIV (20mM Hepes, 1mM EDTA, 0.5mM EGTA) with 5 minutes rotating at room temperature in between. Samples were decrosslinked for 1h at 55°C 1000 rpm shaking followed by an overnight incubation at 65°C using elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris pH8) and proteinase K. Samples were incubated one additional hour the next day with elution buffer and proteinase K for 1 hour at 55°C. The samples were purified using 2x SPRI AMPureXP beads. qPCR was used to determine the sufficient amount of PCR cycles needed to amplify the library. The libraries were amplified using the KAPA HiFi Hotstart Ready Mix (KAPA Biosystems) and Nextera Index Kit 1 (i7) and 2 (i5) primers (Illumina). Amplified libraries were purified using an 0.65× SPRI AMPureXP beads incubation followed by an 1.8× SPRI AMPureXP beads incubation. ATAC-seq was performed as described in (Buenrostro et al, 2015) with three modifications. First, the total volume of the tagmentation reaction was halved. Second, the tagmentation reaction was stopped with 44 mM EDTA, 131 mM NaCl, 0.3% SDS and 600 μg/mL proteinase K. Last, a reverse phase 0.65× SPRI beads (Ampure) DNA purification was done after the first PCR.