title: GSM5918258: mm_islets_10pmFed_Lsd1_ChIPseq_Y; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq: Islets were processed for ChIP immediately following isolation. Mouse islets were hand-picked twice under a dissecting microscope to minimize acinar cell contamination. For histone ChIP, islets were washed once in Hanks Balanced Salt Solution (Hyclone) and then fixed for 10 min in DPBS containing 1.11% formaldehyde. Cross-linking was quenched as before, then islets were lysed by passage through a 25 gauge needle in lysis buffer (10 mM Tris-Hcl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 1% NP-40, 1% SDS, 0.5% sodium deoxycholate). ChIP was then performed as described above using 10 or 30 μg of sheared chromatin and 4 μg of anti-H3K27ac (39133, Active Motif) antibodies, respectively.  For Lsd1 ChIP of islets from fed or fasted mice, 1000 islets from each condition were first dissociated by briefly washing in StemPro Accutase (Life Technologies) and then incubating in StemPro Accutase for at 37 °C for 12 minutes with frequent trituration. Digestion was quenched with HBSS supplemented with 0.3% BSA and 2.8 mM glucose, then islet cells were pelleted via centrifugation for 5 minutes at 1200 rpm in a swing-out rotor centrifuge. Cells were washed briefly in DPBS and then fixed at room temperature on a rocker using 2 mM disuccinimidyl glutarate (Proteochem) for 30 min followed by 1.11% formaldehyde for 15 min. Fixation was quenched with 0.125 M glycine, then cells were washed twice in ice-cold DPBS containing 0.5% BSA. Nuclei were isolated by resuspending cells in lysis buffer (10 mM HEPES-KOH pH 7.9, 85 mM KCl, 1 mM EDTA, 1% NP-40, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF) and incubation for 5 min on ice followed by centrifugation at 1100 x rcf for 8 min at 4°C to pellet nuclei. Nuclear lysis buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.2% sodium deoxycholate, 1% NP-40, 0.5 mM DTT, 1x protease inhibitor cocktail (Sigma), 1 mM PMSF) was then added and chromatin was sheared using a Covaris E220 for 30 cycles with the following settings: Peak power: 140, duty factor: 5, cycles/burst: 200, Avg. Power: 7.0. Immunoprecipitation as performed by overnight rotation at 4°C using 30 μL of Dynabeads Protein A beads (Life Technologies) conjugated to 4 μg anti-Lsd1 antibody (ab17721, Abcam). Beads were then washed using a magnetic rack for 30 s each with the following: 3 washes of RIPA-NR wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% sodium deoxycholate, 1% NP-40, 0.5 mM DTT, 1x protease inhibitor cocktail (Sigma)), 6 washes RIPA-LiCl wash buffer (10 mM Tris-HCl pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40, 1x protease inhibitor cocktail (Sigma)), 3 washes TET buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.2% Tween-20), 1 wash TEN buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl), and 1 wash IDTE buffer (10 mM Tris-HCl pH 8.0, 100 μM EDTA). DNA was then eluted by adding proteinase K (80 μg) and RNase A (40 μg) in reverse crosslinking buffer (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% SDS) and incubating at 50°C for 1 hour then at 65°C overnight. DNA was eluted a second time using 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS and combined with the first elution. The same protocol was followed for Srf ChIP-seq in Min6 cells with the following modifications: cells were harvested using a cell scraper and sonicated using a Covaris E220 for 18 cycles with the above settings, then chromatin from 1.25 x10^7 cells was used for immunoprecipitation using 4 μl of Srf antibody (5147, Cell Signaling). mRNA-seq: Mouse islets were processed for RNA purification immediately following isolation. Following mouse islet isolation, islets were hand-picked twice under a dissecting microscope to minimize acinar contamination, and were then pooled from at least 2 animals per biologicla replicate. For all mRNA-seq experiments, RNA was isolated using the RNeasy Micro kit (QIAGEN) according to the manufacturer's instructions. ChIP-seq ibraries were constructed from purified DNA using the KAPA DNA Library Preparation Kit for Illumina (Kapa Biosystems). Input libraries were prepared from each experimental replicate using 10 ng of DNA purified immediately following shearing. Libraries were sequenced using NovaSeq 6000 (Illumina). mRNA-seq libraries were prepared from 35 ng of total RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina).