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| Status |
Public on Jul 20, 2020 |
| Title |
Primary Schwann Cell cAMP 3 |
| Sample type |
SRA |
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| Source name |
primary Schwann cell
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| Organism |
Rattus norvegicus |
| Characteristics |
genotype: wild type
treatment: CPT-cAMP
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| Treatment protocol |
n/a
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| Growth protocol |
n/a
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Qiagen Rneasy Mini Kit (Catalog Number 74104) with on-column Dnase treatment (Catalog Number 79254) according to manufacturer protocol.
Tn5Prime libraries were constructed essentially as described by Cole and colleagues (NAR, 2018). Briefly, RNA was reverse-transcribed by SMARTScribe RT in the presence of an oligo-dT primer and template-switching oligo. The reaction was treated with RNase and Exonuclease. The resulting DNA was subjected to an indexing PCR, tagmentation by purified Tn5 enzyme pre-loaded with Tn5ME-B/R adapters, and another round of PCR. Size selection to 400-1000bp was performed on a 0.8% low-melt agarose TAE gel. DNA was purified using the Qiagen Gel Extraction Kit (Catalog Number 28704). Libraries were sequenced on Illumina HiSeq or NovaSeq sequencers. Read 1 (corresponding to 5' end of mRNAs) was used for subsequent analyses.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Cells were maintained in medium containing 10% FBS, 10nM neuregulin, and 2uM forskolin, in addition to glutamine, pen-strep, and gentamicin. Cells were plated on poly-L-lysine- and laminin-coated dishes. The next day medium was replaced with medium lacking neuregulin and forskolin. The following day medium was replaced with medium containing 5% FBS. The following day medium was replaced with medium containing 5% FBS and supplemented with 250 uM CPT-cAMP. CPT-cAMP medium was replenished each day for 3 days.
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| Data processing |
Sequenced reads were trimmed of adapter sequences using Cutadapt (v 1.14)
Read 1 from each sample was mapped to the rn5 genome using STAR (v 2.5.2a)
BAM files were generated using Samtools (v 1.9)
Per-base start site counts were generated using the Make_CTSS script from Takahashi and colleagues (Nature Protocols, 2012). TSS clustering was performed using Paraclu (v 9).
Per-cluster read counts were generated using featureCounts (subread v 1.6.1). Statistical analysis was performed using edgeR (v 3.24.3).
Genome_build: rn5
Supplementary_files_format_and_content: Tab-delimited text file includes 4,993 TSSs associated with SOX10-bound promoters in sciatic nerve. For each TSS, genomic coordinates, id number, strand, associated gene symbol, and coordinates of H3K4me3 and SOX10 ChIP-Seq peaks are provided (ChIP-Seq peaks from previously published datasets). Following are the reads per million (RPM) values for each TSS in each sample as measured by Tn5Prime. Statistical values (log2 fold change and FDR-corrected p-values) are provided for cAMP versus control-treated primary Schwann cells and ΔSOX10 versus parental S16 cells.
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| Submission date |
Oct 24, 2019 |
| Last update date |
Jul 20, 2020 |
| Contact name |
Elizabeth Fogarty |
| Organization name |
University of Michigan
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| Street address |
1105 N. University Ave
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL25947 |
| Series (1) |
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| Relations |
| BioSample |
SAMN13107900 |
| SRA |
SRX7051001 |
| 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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