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Sample GSM5918269

Query DataSets for GSM5918269

Status

Public on Feb 24, 2023

Title

mm_islets_CtrlforLsdKI_RNAseq_D

Sample type

SRA

Source name

Pancreatic islets

Organism

Mus musculus

Characteristics

chip antibody: N/A
treatment/condition/replicate: Control for Wk3 Lsd1 KI Rep 4

Growth protocol

Min6 cells were cultured in Dulbecco's modified Eagle's Medium with 25 mM glucose, 4% FBS, 23.8 mM NaHCO3, 1 mM sodium pyruvate, 4 mM L-glutamine, 143 μM β-mercaptoethanol, and 80 μg/mL Gentamicin at 37°C with 5% CO2.

Extracted molecule

total RNA

Extraction 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

ChIP-seq: Bowtie2 v 2.2.7 was used for mapping of ChIP-seq data to the human reference genome hg19 with a seed length of 33 bp and a maximum of 2 mismatches allowed in the seed region, discarding reads aligning to multiple sites. Duplicate reads were removed using SAMtools.
ChIP-seq: Two biological replicates were used for ChIP-seq data analysis, and replicates were merged prior to analysis
ChIP-seq: Peak calling was performed using the findPeaks program of the HOMER suite of bioinformatics tools with the options -style histone, -P 0.0000001 for H3K27ac or -style factor for Lsd1 (with default P-value cutoff of P < 0.0001) with replicate-matched ChIP-seq input used as background.
mRNA-seq: mRNA-seq reads were mapped to NCBI37/mm9 (mouse) or GRCh37/hg19 (human) genomes by STAR (STAR-STAR_2.4.0f1, --outSAMstrandField intronMotif --outFilterMultimapNmax 1 --runThreadN 5), excluding reads mapping to multiple loci. Reads with exact matches were used to determine RPKM by Cufflinks (cufflinks v2.2.1,-p 6 -G $gtf_file --max-bundle-frags 1000000000).
mRNA-seq: Cuffdiff was used to assess expression differences for all pairwise comparisons, with P < 0.01 considered significant. Genes with mean RPKM > 1 in at least one experimental group were considered to be expressed, and all non-expressed genes were excluded from downstream analyses.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-seq: Bedgraph files were generated from merged replicates. Peak files are provided in BED format. mRNA-seq: Cuffdiff output comparing experimental groups

Submission date

Feb 25, 2022

Last update date

Feb 24, 2023

Contact name

Matthew Wortham

E-mail(s)

mwortham@ucsd.edu

Phone

8582460588

Organization name

University of California, San Diego