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| Status |
Public on Oct 11, 2021 |
| Title |
WT-mg-RNA-2_WT RNA REP2 |
| Sample type |
SRA |
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| Source name |
WT RNA
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57/BL6J
tissue: microglia
genotype: WT
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| Extracted molecule |
total RNA |
| Extraction protocol |
Hi-C (Jiang, et. al.): Cortical tissue was prepped for fluorescence activated nuclear sorting (FANS) as follows. Briefly, the dissected tissue was homogenized in a hypotonic lysis solution and fixed in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with 125 mM glycine. The nuclei were then purified by centrifugation at 4000xg and resuspended in a 1:1 solution of the hypotonic lysis solution and a 1.8M sucrose solution prior to re-centrifugation at 4000xg to isolate out cortical nuclei. The pellet was then resuspended in Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% BSA and 1:1000 anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated; EMD Millipore Corp., MAB377X). Samples were incubated for 45 minutes while rotation and protected from light at 4C. DAPI (Invitrogen) was added immediately before sorting to label all nuclei.
ChIP-seq: Cortical tissue was prepped for fluorescence activated nuclear sorting (FANS) as follows. Briefly, the dissected tissue was homogenized in a hypotonic lysis solution and fixed in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with 125 mM glycine. The nuclei were then purified by centrifugation at 4000xg and resuspended in a 1:1 solution of the hypotonic lysis solution and a 1.8M sucrose solution prior to re-centrifugation at 4000xg to isolate out cortical nuclei. The pellet was then resuspended in Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% BSA and 1:1000 anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated; EMD Millipore Corp., MAB377X). Samples were incubated for 45 minutes while rotation and protected from light at 4C. DAPI (Invitrogen) was added immediately before sorting to label all nuclei.
Hi-C (Arima): Cortical tissue was prepped for fluorescence activated nuclear sorting (FANS) as follows. Briefly, the dissected tissue was homogenized in a hypotonic lysis solution and fixed in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with 125 mM glycine. The nuclei were then purified by centrifugation at 4000xg and resuspended in a 1:1 solution of the hypotonic lysis solution and a 1.8M sucrose solution prior to re-centrifugation at 4000xg to isolate out cortical nuclei. The pellet was then resuspended in Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% BSA and 1:1000 anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated; EMD Millipore Corp., MAB377X). Samples were incubated for 45 minutes while rotation and protected from light at 4C. DAPI (Invitrogen) was added immediately before sorting to label all nuclei.
PacBio: Genomic DNA was isolated from FACS-sorted NeuN+ or NeuN- nuclei of adult mouse cerebral cortex using phenol chloroform extraction and was subsequently sheared to ~10kb using g-Tube microcentrifuge tubes (Covaris, 010145).
RNA-Seq (RiboZero): Total RNA was first extracted from the prefrontal mouse cortices (n=12, 6WT/6KO), and prepared with RNeasy Lipid Tissue Mini kit (with on-column DNase1 treatment). The quantity and quality of RNA was checked using a bioanalyzer (Agilent RNA 6000 Nano Kit).
RNA-Seq (QIAseq): Single-cell suspension from adult brain tissues was prepared using Miltenyi’s Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) and Debris Removal Solution Kit (Miltenyi Biotec, 130-109-398) according to manufacturer instruction with minor modifications. In brief, total brain tissues, except olfactory bulb and cerebellum, were collected quickly and washed with ice cold 1x HBSS. After chopped into small pieces using sharp blade, the brain tissues were transferred into the C-tube containing 1950ul of Enzyme mix 1 and 30ul of Enzyme mix 2 from the kit and incubated on Miltenyi’s gentle MACS Octo Dissociator with Heaters using program 37°C _ABDK_1 for 30 minutes. Afterwards, the digested tissue homogenate gently went through fire polished glass pippette 10 times, and then passed through a 70um cell strainer. Cells were collected via centrifuged at 300g for 10 minutes at 4 °C and resuspended in ice cold 1x HBSS. Debris Removal Solution was then added and overlayed with ice cold 1x HBSS. After centrifugation, three phases in the tube were clearly visualized, from top to bottom: 1x HBSS solution, debris and myelin layer, and single-cell suspension. Discard the top two phases completely, wash the cells in 1x HBSS, centrifuge to collect the cells, and then resuspended the cell pellet in 1 ml of 1x HBSS containing 1% fetal bovine serum. In order to minimize the unwanted microglia activation, single cell suspension was incubated in Fc receptor blocking Reagent (Miltenyi Biotec, 130-092- 575) for 10 min, and then the cell suspension was incubated with CD11b-microbeads (Miltenyi Biotec, 130-093-634) for 10 min at 4 °C in the dark, followed by positive selection with LS separation column (Miltenyi Biotec, 130-042-401). Flow cytometry analysis was performed to check the cell purity. The three different groups of cells (no enrichment, target and non-target cell population) were incubated in CD11b-FITC Monoclonal Antibody (M1/70) (eBioscience, 11-0112-81) at 1:2000 dilution for 30 min at 4 °C in the dark. Stained cells were examined using the Beckman flow cytometer.
ATAC-seq: Single-cell suspension from adult brain tissues was prepared using Miltenyi’s Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) and Debris Removal Solution Kit (Miltenyi Biotec, 130-109-398) according to manufacturer instruction with minor modifications. In brief, total brain tissues, except olfactory bulb and cerebellum, were collected quickly and washed with ice cold 1x HBSS. After chopped into small pieces using sharp blade, the brain tissues were transferred into the C-tube containing 1950ul of Enzyme mix 1 and 30ul of Enzyme mix 2 from the kit and incubated on Miltenyi’s gentle MACS Octo Dissociator with Heaters using program 37°C _ABDK_1 for 30 minutes. Afterwards, the digested tissue homogenate gently went through fire polished glass pippette 10 times, and then passed through a 70um cell strainer. Cells were collected via centrifuged at 300g for 10 minutes at 4 °C and resuspended in ice cold 1x HBSS. Debris Removal Solution was then added and overlayed with ice cold 1x HBSS. After centrifugation, three phases in the tube were clearly visualized, from top to bottom: 1x HBSS solution, debris and myelin layer, and single-cell suspension. Discard the top two phases completely, wash the cells in 1x HBSS, centrifuge to collect the cells, and then resuspended the cell pellet in 1 ml of 1x HBSS containing 1% fetal bovine serum. In order to minimize the unwanted microglia activation, single cell suspension was incubated in Fc receptor blocking Reagent (Miltenyi Biotec, 130-092- 575) for 10 min, and then the cell suspension was incubated with CD11b-microbeads (Miltenyi Biotec, 130-093-634) for 10 min at 4 °C in the dark, followed by positive selection with LS separation column (Miltenyi Biotec, 130-042-401). Flow cytometry analysis was performed to check the cell purity. The three different groups of cells (no enrichment, target and non-target cell population) were incubated in CD11b-FITC Monoclonal Antibody (M1/70) (eBioscience, 11-0112-81) at 1:2000 dilution for 30 min at 4 °C in the dark. Stained cells were examined using the Beckman flow cytometer.
Hi-C (Jiang, et. al.): Nuclei were digested with 100U MboI, and the restriction fragment ends were labeled using biotinylated nucleotides and re-ligated. After reversal of the cross-linking, ligated DNA was purified and sheared to a length of ~400bp by sonication, and the biotin-tagged ligation junctions were subsequently pulled down with streptavidin beads (Invitrogen, Dynabeads MyOne Streptavidin T1, Catalog No. 65602) and prepared into libraries.
ChIP-seq: Nuclei were pelleted after FANS, resuspended in 300μl of micrococcal nuclease digestion buffer (10mM Tris pH 7.5, 4mM
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Description |
QIAseq
WT-mg-RNA-2
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| Data processing |
Hi-C (.hic files):
Quality control was performed using the ‘pre-truncation method’ with HiC-Pro(v2.9).
Libraries were mapped to the Mus musculus reference genome (GRCm38.p5_M13) using bowtie2.2, and combined by matching sex and genotype.
HiC-Pro results were piped to Juicer(v1.14.8) to produce .hic format files using the parser tool ‘hicpro2juicebox’.
Resulting files were then processed into final KR matrix-balanced normalized contact maps with Juicebox.
Genome_build: GRCm38.p5_M13 or mm10
Supplementary_files_format_and_content: .hic files for Juicebox visualization and subcompartment calling
Hi-C (HOMER interactions):
Each read of the paired-end libraries was aligned independently using bwa-mem(v0.7.15), permitting split mapping to the GRCm38.p5_M13 reference annotation.
After mapping, forward and reverse reads were directly supplied to Homer(v4.8) for processing by first, merging the paired-end reads and later, filtering out self-ligation (spikes and continuous) artifacts.
Files were normalized based on sequencing depth and distance between loci, creating a background model necessary for calculating significant pairwise interactions.
Trans- interactions (1Mb resolution) were similarly calculated, but for significant interactions for loci >200Mbp apart (-minDist 200000000) to disregard intra-chromosomal interactions.
Supplementary_files_format_and_content: HOMER interactions (1Mb resolution, p<1e-50)
differential bins file (NeuN+ vs. NeuN-):
Raw .fastq reads were aligned to the Mus musculus reference genome (mm10) using bowtie2.
Only concordant reads were compressed into aligned .bam files, then sorted and indexed using the samtools/0.1.19 suite.
Following PCR duplicate removal, files were converted into .bed files using bedtools(v2.29.2)for further processing.
Differential analysis using diffReps was performed (1kb resolution) at a statistical significance threshold of p<0.001, for NeuN+ (tx) vs. NeuN- (ctrl).
Significantly called regions (spanning a length of 1kb or more) were labeled as NeuN+ enriched or NeuN- enriched according to their log2FoldChange values.
Supplementary_files_format_and_content: differential bins file (NeuN+ vs. NeuN-)
.txt files with BLAST scores for de novo alignments to the IAP-masked genome or IAPEzi:
Bioinformatics processing was performed with SMRT Link v5.1.0, run with default parameters, unless otherwise indicated.
Files were first demultiplexed with lima and circular consensus sequences (CCS) were generated with ccs for filtered sequences with matching paired-end adapters.
CCS reads were then mapped to an IAPEzi consensus sequence (dfam.org) using pbalign.
Passing reads were subsequently mapped to the mouse reference genes using bwa split mapping and filtered based on mapping score (=60).
Reads were then assigned as autosomal IAPEzi or autosomal non-IAPEzi IAP using the GenomicRanges subsetByOverlaps() function (R(v3.6.0)).
The other reads, those not mapping to IAPs denoted in the reference genome, were then blasted against the IAPEzi consensus sequence and IAP masked reference genome in parallel; reads with higher bit scores for the IAPEzi blast than the reference genome were defined as de novo. Full-length de novo insertions were defined as fragments representing >5833bp (0.9*6481).
Supplementary_files_format_and_content: .txt files with BLAST scores for de novo alignments to the IAP-masked genome or IAPEzi
.txt files with transcript alignments to genes and repeats:
Read pairs were aligned to the Mus musculus mouse reference genome (mm10) with Tophat2 short-read aligner.
Reads were counted using HTSeq against the Gencode vM4 Mouse annotation.
Genes were filtered based on the criteria that all replicates in either condition must have at least 5 reads per gene.
On the resulting filtered transcripts, a pairwise differential analysis between Setdb1 conditional mutant vs. control cortex was performed using the voomlimma R package, which converts counts into precision weighted log2 counts per million and determines differentially expressed genes using a linear model.
Significantly differentially expressed genes were identified using a cutoff of Benjamini-Hochberg adjusted p-value less than 0.05.
Discordant pairs were extracted from aligned .bam files using samtools with the following command: samtools view –b –F 2 and reads identifiable with specific flags were quantified and categorized.
Supplementary_files_format_and_content: .txt files with transcript alignments to genes and repeats
RNA-seq:
FastQC was first used for quality control analysis, and trim-galore was used to remove low-quality reads.
Paired-end cleaned data was aligned to reference genome (M. musculus, UCSC mm10) using Tophat2 v2.1.1.
Samtools v1.9 was used to sort and build the alignment files index.
FeatureCounts v1.6.3 from subread package was used to get gene expression level counts by determining the number of reads mapped to gene exons.
Differential analysis was generated by DESeq2.
Significant genes (log2FoldChange > 0.5, Padj < 0.05) were extracted for Gene Ontology Enrichment Analysis by using ShinyGO v0.61 (http://bioinformatics.sdstate.edu/go/).
Deeptools was used to generate normalized bigwig and visualized on IGV.
Supplementary_files_format_and_content: .txt and .xls counts files
peaks files:
FastQC was first used for quality control analysis, and trim-galore was used to remove low-quality reads.
Paired-end cleaned data was then aligned to reference genome (M. musculus, UCSC mm10) using Bowtie2 v2.3.4.3.
Samtools v1.9 was used to sort and build the alignment files index after removing duplications.
MACS2 was used for peak calling (--shift -100 --extsize 200 --nomodel).
Diffbind was used to generated differential change peaks. R packages “ChIPseeker”, “clusterProfiler” “org.Mm.eg.db” and “TxDb.Mmusculus.UCSC.mm10.knownGene” were used for peak annotation.
Significant differential peaks that are located on gene promoters were extracted to generate Gene Ontology Enrichment Analysis by ShinyGO v0.61 (http://bioinformatics.sdstate.edu/go/).
Deeptools was used to generate normalized bigwig and visualized on IGV.
Supplementary_files_format_and_content: .xls peaks files
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| Submission date |
Mar 09, 2021 |
| Last update date |
Oct 11, 2021 |
| Contact name |
Sandhya Chandrasekaran |
| E-mail(s) |
sandhya.chandrasekaran@icahn.mssm.edu
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| Phone |
5104568992
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Department |
Psychiatry
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| Lab |
Akbarian
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| Street address |
1470 Madison Avenue, 9-202, Hess Building
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE168524 |
NEURON-SPECIFIC CHROMOSOMAL MEGADOMAIN ORGANIZATION IS ADAPTIVE TO RECENT RETROTRANSPOSON EXPANSIONS |
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| Relations |
| BioSample |
SAMN18221456 |
| SRA |
SRX10291365 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5145472_2132wt_microglia_FRBL202378495-1a.total.count.txt.gz |
84.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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