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Sample GSM6210108

Query DataSets for GSM6210108

Status

Public on Jun 14, 2022

Title

PRMT1Ifl/fl microglia normal diet_Input_rep2

Sample type

SRA

Source name

C57BL/6

Organism

Mus musculus

Characteristics

strain: C57BL/6
cell type: Microglia
genotype: PRMT1Ifl/f
treatment: Normal diet
time: 11 weeks
chip antibody: none (input)

Treatment protocol

0.2% of Cuprizone diet was fed in 4-6 weeks-old wild-type (PRMT1Ifl/fl) and microglia specific knockout (PRMT1fl/fl; cx3r1cre-ert2) mice
Brains were digested, and myelin were removed using 30% percoll gradient. Microglia was sorted using CD45 and CD11B surface markers. RNA in 2X SuperScript III buffer was incubated for 1 min at 50 C on a PCR cycler with 2.5 ul of the following mix: 1.5 ug Random Primer (Life Technologies 48190-011), 10 M Oligo d(T) (Life Technologies 18418020), 10 units SUPERase-In (Ambion AM2696), 4 mM dNTP mix (Life Technologies 18427088), in water. Samples were then immediately placed on ice for 5 min. First-strand synthesis was then performed by incubation at 25 C for 10 min and 50 C for 50 min on a PCR cycler with 7.6 ul of the following mix: 0.2 ug actinomycin (Sigma A1410), 13.15 mM DTT (Life Technologies), 0.026% Tween-20, 100 units SuperScript III (Life Technologies kit 18080-044), in water. After incubation, RNA/DNA complexes were isolated by adding 36 ul of Agencourt RNAClean XP beads (Beckman Coulter A63987) and incubated for 10 min at RT and then 10 min on ice. Samples were then placed on a magnet and beads were washed twice with 150 ul of 75% EtOH. Following washings, beads were air-dried for 10-12 min and eluted with 10 ul of water. Second-strand synthesis was then performed. RNA/DNA samples in 10 ul of water were incubated for 2.5 hours at 16 C with 5 ul of the following mix: 3x Blue Buffer (Enzymatics), 1.0 ul PCR mix (Affymetrix 77330), 2.0 mM dUTP (Affymetrix 77206), 1 unit RNAseH (Ezymatics Y9220L), 10 units DNA Polymerase I (Enzymatics P7050L), 0.03% Tween-20, in water). DNA was then purified by addition of 1.5 ul Sera-Mag SpeedBeads (Thermo Scientific, 651520505025), resuspended in 30 ul 20% PEG 8000/2.5 M NaCl, incubated at RT for 15 min and placed on a magnet for two rounds of bead washing with 80% EtOH. Beads were then air dried for 10-12 min and DNA was eluted from the beads by adding 40 ul of water. Supernatant was then collected on a magnet and placed on ice or stored at -20C until DNA blunting, poly(A)-tailing, and adapter ligation.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin immunoprecipitation for histone modification H3K27ac and was performed as follows, using ~500,000 microglia per assay and n=2 independent biological replicates per conditions. First microglia were briefly thawed on ice and lysed by incubation in 1 ml of lysis buffer (0.5% IGEPAL CA-630, 10 mM HEPES pH 7.9, 85 mM KCl, 1 mM EDTA pH 8.0, in water) for 10 min on ice. Lysates were centrifuged at 800 RCF for 5 min at 4 °C, and pellets were resuspended in 200ul of sonication/ immunoprecipitation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 0.1% Deoxcycholate, 0.5% Sarkosyl, in water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 15 min each, alternating stages of 30 sec “sonication-on” with 60 sec “sonication-off”. Twenty-two microliters of 10% Triton-X were then added to samples (1% final concentration) on ice, and lysates were cleared by centrifugation for 5 min at 18,000 RCF at 4 °C. Two microliters of supernatant were then set aside for input library sequencing controls. Supernatants were then immunoprecipitated on a rotator for two hours at 4 °C with H3K27ac antibody (Active Motif, 39685, 2.5 ug per sample) pre-bound to 17 ul Protein A Dynabeads (Life Technologies 10001D). Immunoprecipitates were washed three times each on ice with ice-cold wash buffer I (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA pH 8.0 in water), wash buffer III (10 mM Tris-HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1mM EDTA in water) and TET (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.1% Tween-20, in water) and eluted with 1% SDS/TE at RT in a final volume of 100 ul. Reverse-crosslinking was then performed on immunoprecipitates and saved input aliquots. First, 6.38 ul of 5 M NaCl (final concentration 300 mM) was added to each sample immersed in 100 ul SDS/TE. Crosslinking was then reversed by overnight incubation at 65 °C in a hot air oven. Potentially contaminated RNA was then digested for one hour at 37 °C with 0.33 mg/ml RNase A, proteins were digested for one hour at 55 °C with 0.5 mg/ml proteinase K, and DNA was extracted using Sera-Mag SpeedBeads (Thermo Scientific, 6515205050250).
Sequencing libraries were prepared from recovered DNA (ChIP) or generated cDNA (RNA) by blunting, A-tailing, and adapter ligation as previously described using barcoded adapters (NextFlex, Bioo Scientific) (Gosselin et al., 2014; Gosselin et al., 2017). Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-400 bp for ChIP-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). ChIP-seq librairies were single-end sequenced for 76 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA) according to manufacturer’s instruction.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

ChIP

Data processing

Single-end reads of length 76 were first trimmed off the adapter sequence as well as the polyA tail and then mapped through the Bowtie2 short read aligner v2.4.1 to the hg19 reference genome from the University of California Santa Cruz (UCSC) (Lander et al., 2001; Langmead and Salzberg, 2012).
Quality control checks on the raw sequence data were carried out through the FASTQC software v0.11.9. BAM files were sorted and indexed, and duplicate reads were removed through Samtools v0.1.19.
Peak calling and motif enrichment for ChIP-seq experiments relative to the input was carried out by the Hypergeometric Optimization of Motif Enrichment (HOMER) software v4.11 in histone or super-enhancer mode using default parameters(Heinz et al., 2010).
Average read coverage profiles across TSS, TTS or gene body regions and corresponding heatmaps were plotted with the NGS PLOT software (Shen et al., 2014). Density plots of reading coverage were made using the Integrative Genomics Viewer (IGV) software.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include normalized tage counts for each Sample

Submission date

Jun 01, 2022

Last update date

Jun 14, 2022

Contact name

Stephane Richard

E-mail(s)

[email protected]

Phone

5143408260

Organization name

Lady Davis Institute- Jewish General Hospital