title: NextSeq 500 sequencing; HNF1A, UTX, H3K27me3 and H3K27ac ChIP-seq in wild-type and UTX- or HNF1A-deficient mouse pancreas
design: HNF1A, UTX, H3K27me3 and H3K27ac ChIP-seq in wild-type and UTX- or HNF1A-deficient mouse pancreas
library_name: Input_cKO_s
library_construction_protocol: ChIP experiments were performed on pancreas from day 4 old or 3 weeks old female mice. For day 4 samples, 2 pancreas were processed per UTX ChIP and 8 pancreas per HNF1A ChIP. For 3 weeks samples, one pancreas were processed per ChIP. A Polytron was used to quickly mince dissected pancreas in PBS with protease inhibitors followed by crosslinking with 1% formaldehyde for 10min at room temperature on a rotator and then quenched with 0.125M glycine for 5min. Tissue pieces were washed twice in PBS and lysed in 130 µl lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) with protease inhibitor on ice for 10 min and finally resuspended with a 30G needle syringe. The chromatin preparation was then sonicated to fragments enriched in the size range of 150 to 500 bp using S220 Focused Ultrasonicator (Covaris) and centrifuged at 14000 rpm for 10 min at 4°C to pellet insoluble material. The supernatant was incubated for 1 h with 350 µl RIPA-LS buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1mM EDTA, pH 8.0, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100), protease inhibitors, and 20 µl Dynabeads Protein G for pre-clearing. After preserving 1% as an input sample the pre-cleared chromatin was incubated with 2 µg antibody, 50 µl 10% BSA and 5 µl tRNA (10 mg/ml) overnight at 4°C with rotation. Immune complexes were retrieved with 20 µl BSA blocked Dynabeads Protein G for 2 h at 4C and washed 2x with ice cold RIPA-LS, 2x with RIPA-HS (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1mM EDTA, pH 8.0, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100), 2x with RIPA-LiCl (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1mM EDTA, pH 8.0, 0.5% IGEPAL, 0.5% Na-Deoxycholate) and 1x with 10 mM Tris, pH 8.0 ChIP DNA was purified with Qiagen MinElute kit (QIAGEN) and enrichment cycles for library amplification was assessed by qPCR. Initial steps of library preparation introduces sequencing adapters by tagmentation directly on the bead-bound chromatin according to a ChIPmentation procedure that is optimized for low cell number samples (Schmidl et al., 2015). Briefly, chromatin bound beads were resuspended in 25 μl of tagmentation reaction buffer (10 mM Tris, pH 8.0, 5 mM MgCl2, 10% v/v dimethylformamide) containing 1 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) and incubated at 37°C for 10 min followed by 2x washing in RIPA-LS and TE (10mM Tris-HCL, pH 8.0, 1mM EDTA, pH 8.0). The DNA was then de-crosslinked and eluted from the beads in ChIP elution buffer (10mM Tris-HCL, pH 8.0, 5mM EDTA, pH 8.0, 300mM NaCl, 0.4% SDS) with proteinase K at 55°C for 1 h and 65°C overnight. The libraries were PCR amplified with KAPA HiFi Hotstart Ready mix (Sigma-Aldrich) and barcoded Nextera custom primers and finally size-selected (250–350 bp) using Agencourt AMPure XP beads (Beckman Coulter) and validated using the Agilent High Sensitivity DNA Kit with Agilent 2100 Bioanalyzer.
attributes.tag: Experimental Factor: genotype
attributes.value: Pdx1Cre;Utx LoxP/LoxP
attributes.tag: Experimental Factor: age
attributes.value: 4
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA