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| Status |
Public on Jun 30, 2021 |
| Title |
Vehicle Treated, Replicate 5 |
| Sample type |
SRA |
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| Source name |
Cell line
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| Organism |
Mus musculus |
| Characteristics |
cell line: N2A
treatment: vehicle (100% DMSO)
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| Treatment protocol |
Basal medium (2% FBS in DMEM), with 1% penicillin-streptomycin in the medium before transfection and >6hrs after plus 20µM final concentration all-trans retinoic acid in 100% DMSO, or an equivalent amount of 100% DMSO for vehicle treatment. Medium prepared and refreshed daily with freshly added ATRA from light-protected frozen aliquots
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| Growth protocol |
Cells were grown without changing medium for 72 hours at 37°C, 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells lifted in 250uL PBS and 750µL Trizol, followed by addition of 200uL chloroform and cleanup of the aqueous phase using the Zymo Clean and Concentrator 5 kit per manufacturer instructions. Turbo DNAse-free kit was used to remove incidental DNA, followed by a second cleanup using the Zymo kit to achieve a higher final concentration of RNA.
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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| 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.
For each allelic sequence pair (each allele having up to 10 unique RNA barcodes), mean of the rna/dna ratios from above calculated for each sample --> t-test of sample mean expression values between the alleles of each biallelic 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 |
Feb 25, 2021 |
| Last update date |
Jun 30, 2021 |
| 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) |
| GSE167518 |
Transcriptional-Regulatory Convergence Across Functional MDD Risk Variants Identified by Massively Parallel Reporter Assays [Expt2] |
| GSE167519 |
Transcriptional-Regulatory Convergence Across Functional MDD Risk Variants Identified by Massively Parallel Reporter Assays |
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| Relations |
| BioSample |
SAMN18056908 |
| SRA |
SRX10168405 |
| 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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