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Sample GSM3978627

Query DataSets for GSM3978627

Status

Public on Feb 24, 2023

Title

mm_islets_d7Ctrl_input_ChIPseq_B

Sample type

SRA

Source name

Pancreatic islets

Organism

Mus musculus

Characteristics

chip antibody: none
treatment/condition/replicate: Control for Wk1 Lsd1 KO Rep 2

Treatment protocol

Human islet mRNA-seq: Upon receipt, human islets were stained with dithizone and positively-staining material was hand-picked using a dissection microscope. Islets were allowed to recover 18-48 hours in complete media (CMRL 1066 with 13 mM glucose, 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, 10 mM HEPES, and 0.25 mg/mL amphoterecin B) at 37°C with 5% CO2 prior to treatments. For LSD1 inhibition studies, 2 mM tranylcypromine (Sigma) was included in the culture media for 24 hours prior to analysis.

Extracted molecule

genomic DNA

Extraction protocol

ChIP-seq: Islets were processed for ChIP immediately following isolation (for mouse islets) or shipping (for human islets). Mouse islets were hand-picked twice under a dissecting microscope to minimize acinar cell contamination. Human islets selected for ChIP-seq analysis were of > 90% purity to facilitate analysis of large quantities of islets without the need for dithizone staining. For Lsd1 ChIP, islets were transferred to complete islet media (CMRL 1066 with 13 mM glucose, 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, 10 mM HEPES, and 0.25 mg/mL amphoterecin B) containing 1.11% formaldehyde and fixed on a rocker for 15 minutes. The reaction was quenched for 5 minutes in 0.125 M glycine. Cells were then washed in DPBS containing 0.5% NP-40 then once again in DPBS supplemented with 0.5% NP-40 and 1 mM PMSF. Islets were lysed and ChIP was performed using the ChIP-IT High Sensitivity Kit (Active Motif) with 30 μg of sheared chromatin and 4 μg anti-Lsd1 antibody (ab17721, Abcam). 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-H3K4me1 (ab8895, Abcam) or anti-H3K27ac (39133, Active Motif) antibodies, respectively. mRNA-seq: Human islets were first treated as described under the "treatment protocol" section above, while 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 HiSeq 4000 (Illumina). mRNA-seq libraries were prepared from 35 ng of total RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina) with the exception of db/db mouse islets and their respective controls, from which libraries were generated with 1 μg of total RNA as described in (Xie, R. et al. Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells. Cell Stem Cell 12, 224-237, 2013).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

ctrl_d7input.ucsc.bedGraph.gz

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 or hg19
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

Jul 25, 2019

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