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| Status |
Public on Apr 18, 2019 |
| Title |
whole-cell PASseq rep2 |
| Sample type |
SRA |
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| Source name |
whole-cell PASseq
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| Organism |
Rattus norvegicus |
| Characteristics |
tissue source: Embryonic day 19 rat hippocampus
cell type: Primary hippocampal neurons
days in vitro: 14
cell culture platform: Semipermeable membrane with 1 μm holes
cellular fraction: Whole-cells
molecule subtype: polyA RNA
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| Growth protocol |
Hippocampal neurons were extracted from embryos of pregnant rats at 19 days of gestation and maintained in culture for 14 days. To separate projections from cell bodies, neurons were cultured on poly-L-lysine coated semipermeable membranes with 1 μm holes. For cytoplasmic fractionation, neurons were cultured on 6-cm poly-L-lysine coated, standard tissue culture plates. Cells were maintained in Neurobasal media (Invitrogen 21102-049), supplemented with glutamax (Gibco™ 35050061), antibiotic/antimycotic (Gibco™ 15240062), and B27 (Gibco™ 17504001). On day three, Cytosine beta-D-arabinofuranoside (Sigma-Aldrich #C1768), a DNA replication inhibitor, was added to the media to inhibit glial cell division. On day six and day twelve of neuron culture, half of the Neurobasal media was replaced with fresh media. On day 14, cells were harvested for sequencing library preparation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Library preparation method: Ashar-Patel A., … Moore M.J. (Scientific Reports, 2017).
RNA extraction: For neurons cultured on semipermeable membranes, RNA was extracted following the steps recommended by TRIzol® reagent (ThermoFisher #15596018) manual with minor modifications. Briefly, media was removed and TRIzol® was applied to the projections-side, the surface was scraped using a cell-scraper and lysate was gently pippeted and transferred into a clean tube. Lysate was similarly collected from the cell body side of the membrane. The lysate was vortexed briefly and kept at room temperature for 5 minutes. 200 μl of chloroform per 1 ml of TRIzol® was added for phase separation. The tubes were vigorously shaken by hand and centrifuged at 12,000 x g for 15 min at 4°C. The aqueous phase was transferred to fresh tubes and the chloroform wash was repeated two more times. RNA was precipitated by adding 100% isopropanol. Samples were incubated at room temperature for 15 min, and centrifuged at 12,000 x g at 4°C for 15 min. The supernatant was removed without disturbing the precipitated RNA. The RNA precipitate was rinsed two times 75% ethanol and finally dissolved in RNase-free water by incubating for 10 min at 55-60°C.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Genome build: rnor6
Reads were trimmed using cutadapt version 1.7.1 to remove a 7nt barcode sequence from the 5' end and stretches of As from the 3' end. The reads were further selected for > 35 quality and minimum 25 nt length. Filtered reads were aligned to rnor6 genome using the following TopHat2 parameters: $ tophat/2.1.1/tophat -p 4 --library-type fr-secondstrand --b2-very-sensitive --no-novel-juncs -i 30 -g 10 -G <genes.gtf> -o <output_directory> <genome_index_base> <PASseq_reads.fastq>. PolyA sites were identified using CleanUpdTseq (Sheppard et al., 2013), and read coverage on statistically significant polyA sites is provided as a bigwig file.
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| Submission date |
Apr 16, 2019 |
| Last update date |
Apr 18, 2019 |
| Contact name |
Harleen Saini |
| E-mail(s) |
harleen_saini@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Lab |
Danesh Moazed
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| Street address |
240 Longwood Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL20084 |
| Series (1) |
| GSE129924 |
Free circular introns with an unusual branchpoint in neuronal projections |
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| Relations |
| BioSample |
SAMN11444435 |
| SRA |
SRX5701010 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3728203_WhCell_PASseq_rep2.bigwig |
706.5 Kb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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