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| Status |
Public on Feb 10, 2023 |
| Title |
M_Hippocamp_2 |
| Sample type |
SRA |
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| Source name |
Hippocampus
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| Organism |
Mus musculus |
| Characteristics |
tissue (trap fraction): Total hippocampus
material: RNA
Sex: M
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| Treatment protocol |
Stereotactic delivery of AAV9 to 1 hippocampal coordinate at 2 depths per side, 2µL of AAV9 per site (4 sites/mouse) 28 days prior to tissue collection.
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| Growth protocol |
Ad libitum feeding in single-sex group housing on 12 hour light/dark cycle.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Hippocampus dissected away from rest of brain, homogenized with power drill in glass pestle with RNAse inhibitors and translation inhibitor cycloheximide, spun 2,000g, supernatant mixed with 10% NPC and 10% DHPC 20min on ice, 20,000g spin, part of supernatant added to trizol for "input" (total hippocampal) RNA and remainder used for immunoprecipitation of GFP+ ribosomes (i.e. TRAP). See methods in paper for full details.
Reporter-specific cDNA synthesis using a reverse-complementary primer, followed by PCR, digestion, adapter ligation, and index PCR. DNA from plasmid was prepared simultaneously, starting with the first PCR step. Each step was followed by a cleanup using size-selection beads (brands Ampure XP and Magbind).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
M_Pre2
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| Data processing |
Barcode extraction from sample fastq - see bitbucket repository for script. Requires flanking 14 bp around barcode to be identical to design, plus a 10bp sequence to be found in between the flanks. All 10bp sequences extracted from Fastqs into text file.
Tabulation of barcode counts for each sample using table() in R. Subsequently, merging to a metadata table on designed barcodes to drop sequences that were not a perfect match to a designed barcode.
Filtering for low read counts, poor barcode representation across replicates, etc. Described at length in Supplementary Methods.
Calculation of log2(cpm RNA barcode) / log2(cpm DNA barcode) for each retained barcode in each sample.
Subtract the within-sample mean of "basal" (minimal-promoter-only) barcode expression from all other barcode expression values --> REML linear mixed model: expression ~ allele + sex + sex:allele + (1|BC), where BC is the unique barcode identifier to control for effects of barcode sequence.
Genome_build: hg19-derived sequences (up to 126bp); see enclosed file "Reference-Barcode Sequences, their paired internal identifiers, and the corresponding sequence placed upstream of min promoter.txt" for each allele of each sequence and its paired reporter (RNA) barcodes
Supplementary_files_format_and_content: Tab-delimited text; barcode counts for each RNA sample and the DNA sample.
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| Submission date |
Oct 21, 2021 |
| Last update date |
Feb 10, 2023 |
| Contact name |
Joseph Dougherty |
| E-mail(s) |
mulveyb@wustl.edu, jdougherty@wustl.edu, berniejmulvey@gmail.com
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| Organization name |
Washington University in St. Louis
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| Department |
Genetics
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| Lab |
Dougherty
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| Street address |
660 S Euclid Ave, Campus Box 8232
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE186346 |
Extensive sex differences in depression-linked variants functionally assayed in mouse brain [Hippocampus] |
| GSE186348 |
Extensive sex differences in depression-linked variants functionally assayed in mouse brain |
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| Relations |
| BioSample |
SAMN22486674 |
| SRA |
SRX12727603 |
| 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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