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| Status |
Public on Jul 01, 2022 |
| Title |
MG_MACS_CON_P4_Rep5 |
| Sample type |
SRA |
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| Source name |
whole brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: whole brain
cell type: microglia
group: CON
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| Extracted molecule |
total RNA |
| Extraction protocol |
P4 mice were not perfused but blood was drained by decapitation, and brains extracted. The meninges were removed and the whole brain collected into 2 mL tubes with HBSS on ice. The brain pieces were diced with scissors and then homogenized with papain according to the adult neural dissociation kit (Miltenyi). Briefly, the tissue was treated with papain and DNase at 37°C for 30 min with rotation. The homogenates were pipetted gently and filtered with a 70 µm strainer. The cell pellet was resuspended in debris removal solution (300 µL debris removal, 700 µL PBS), overlaid with 300 µL of PBS, centrifuged at 3000xg for 10 min, debris aspirated, washed with PBS, and centrifuged at 1000xg for 10 min. The cells were resuspended in 0.5% BSA in HBSS. For magnetic sorting anti-CD11b magnetic beads were added for 30 min at 4C. The cells were washed with buffer and isolated on the magnetic column before final collection into RNA lysis buffer. RNA was collected according to the kit instructions (Qiagen, Micro-RNeasy, PicoPure RNA isolation Kits).
Bulk cell RNA sequencing libraries were prepared using a modified Smart-Seq2 protocol. 5 ng of purified RNA were mixed with 2.5 mM dNTP mix, 2.5 µM oligo-dT30VN, and 1 U/µL RNasin and incubated at 72°C for 3 min. Then 5.7 µL of the single-cell reverse transcription mix (1x superscript II buffer, 1 M betaine, 5 mM DTT, 100 U superscript II, 10 U RNasin, 1 µM TSO oligo, 6 mM MgCl2) was added to each sample for reverse transcription. The cDNA was amplified with the KAPA HF Ready mix with 9 PCR cycles. The cDNA was cleaned up with AMPure XP beads (Beckman Coulter) at a ratio of 0.7:1, washed with 80% ethanol, and resuspended in 17.5 µL water. The libraries were tagmented for sequencing using the Illumina Nextera XT DNA sample preparation kit. 200-250 pg of cDNA was added to 2.5 µL tagment DNA buffer (Illumina) and 1.25 µL of amplicon tagment mix (Illumina) and incubated at 55°C for 5 min to tagment cDNA. The reaction was neutralized with 1.25 µL of neutralize tagment buffer and incubated at room temperature for 5 min. Then the tagmented DNA was indexed and amplified with 3.75 µL of PCR master mix (Illumina) and 1.25 µL each of i5 and i7 indexing primers (Illumina, diluted 1:5). The samples were indexed with the following PCR cycles: 1 cycle of 72°C for 3 min and 95°C for 30 sec; 12 cycles of 95°C for 10 sec, 55°C for 30 sec, and 72°C for 30sec, and 1 cycle of 72°C for 5 min. The final libraries were purified using AMPure XP beads (Beckman Coulter) at a 0.6:1 ratio, washed with 80% ethanol and resuspended in 12 µL water.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
CS5
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| Data processing |
Alignment: FASTQ files aligned and assembled with RSEM and STAR: rsem-calculate-expression -p 4 --paired-end --star --star-output-genome-bam --calc-ci --forward-prob 0.5
Counts: Count matrix was assembled using tximport in R files <- list.files(path = "../1.align/genes.results/") names(files) <- strtrim(files, 4) txi.rsem <- tximport(files, type = "rsem", txIn = FALSE, txOut = FALSE) txi.rsem$length[txi.rsem$length == 0] <- 1 table(txi.rsem$length == 0) save(txi.rsem, file = "txi.rsem.RData")
Differential expression was performed with DESeq2.
Assembly: GRCm38vM25
Supplementary files format and content: Neonatal_MACS_counts.csv: Comma-separated file of raw counts from RSEM output.
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| Submission date |
Apr 28, 2022 |
| Last update date |
Jul 01, 2022 |
| Contact name |
Lindsay N Hayes |
| E-mail(s) |
lhayes14@jhmi.edu
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| Organization name |
Johns Hopkins University
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| Department |
Neuroscience
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| Street address |
600 North Wolfe Street, Meyer 4-136
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21287 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE201807 |
Prenatal immune stress blunts microglia reactivity which impairs neurocircuitry [Neonatal_MACS] |
| GSE201817 |
Prenatal immune stress blunts microglia reactivity which impairs neurocircuitry |
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| Relations |
| BioSample |
SAMN27959258 |
| SRA |
SRX15041097 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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