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| Status |
Public on Jul 01, 2023 |
| Title |
C/B TSC NTG-gRNA-Cas9 clone 1 RNA |
| Sample type |
SRA |
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| Source name |
F1-hybrid C/B trophoblast stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: F1-hybrid C/B trophoblast stem cells
strain: CAST/EiJ mother X C57BL/6J father
passage number: 25-30
genotype: NTG CRISPR-deletion
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| Growth protocol |
TSCs were cultured as in (Quinn et al. 2006). Briefly, TSCs were cultured on gelatin-coated, pre-plated irradiated mouse embryonic fibroblast (irMEF) feeder cells in TSC media (RPMI [Gibco, cat #: 11875093], 20% qualified FBS [Gibco, cat #: 26140079], 0.1mM penicillin-streptomycin [Gibco, cat #: 15140122], 1mM sodium pyruvate [Gibco, cat #: 11360070], 2mM L-glutamine [Gibco, cat #: 25030081], 100uM beta-mercaptoethanol [Sigma-Aldrich, cat #: 63689]) supplemented with 25ng/mL FGF4 (Gibco, cat #: PHG0154) and 1ug/mL Heparin (Sigma-Aldrich, cat #: H3149) just before use, at 37°C in a humidified incubator at 5% CO2. At passage, TSCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution (Gibco, cat #: 25200-072) for ~4 minutes at room temperature and gently dislodged from the plate with a sterile, cotton-plugged Pasteur pipette. To deplete irMEFs from TSCs prior to all harvests, TSCs were pre-plated for 45 minutes at 37°C, transferred to a fresh culture plate, and then cultured for three days in 70% irMEF-conditioned TSC media supplemented with growth factors as above. For RNA isolation, TSCs were passaged once off of irMEFs as above onto a single well of a 6-well plate.
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| Extracted molecule |
total RNA |
| Extraction protocol |
ESCs were grown to >75% confluency prior to RNA harvest using 1mL TRIzol, followed by the addition of 200uL chloforom, which were vortexed and subsequently spun at max speed for 5 minutes at 4°C for phase separation. The aqueous layer was collected and combined with 1 volume of 100% isopropanol and 5uL linear acrylamide. Precipitation was achieved at -80°C for 1 hour, followed by a max speed spin for 30 minuttes at 4°C and one wash of the RNA pellet with ice-cold 80% ethanol. The pellet was then resuspended in 100uL pico H2O and quantified via Qubit (Invitrogen, cat #: Q32855).
RNA-Seq libraries were prepared from 1ug of total RNA using KAPA RNA HyperPrep Kit with Ribose Erase (Kapa Biosystems, cat #: KR1351) according to the manufacturer instructions. Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit on a NextSeq 500 System.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Ribo-depleted RNA
ntg_avg_rna_negative.wig.gz
ntg_avg_rna_positive.wig.gz
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| Data processing |
RNA-Seq reads were aligned using STAR with default parameters (Dobin et al. 2013).
For all RNA-Seq analyses in this study, reads that had a mapping quality greater than or equal to 30 were extracted with Samtools, and allele-specific read retention (i.e., reads that overlap at least one B6 or CAST SNP) was performed as in (Calabrese et al. 2012; Calabrese et al. 2015; Schertzer et al. 2019a) using a custom perl script (intersect_reads_snps16.pl: see github).
Strand-specific reads were separated using samtools: samtools view -Sq 30 (negative) -F 0x10 or (positive) -f 0x10
Assembly: All mouse reference NCBI build 37/mm9 genome annotations were obtained from the UCSC genome browser (Lee et al. 2022). Variant sequence data was obtained from the Sanger Institute (http://www.sanger.ac.uk/resources/mouse/genomes/; (Keane et al. 2011). The CAST/EiJ (CAST) pseudogenome creation was performed as in (Calabrese et al. 2012; Calabrese et al. 2015; Schertzer et al. 2019a).
Supplementary files format and content: Wiggle density files were created using a custom perl script (bigbowtie_to_wig3_mm9.pl; see github). The WIG file includes reads pooled from the two or more biological clones.
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| Submission date |
Nov 04, 2022 |
| Last update date |
Jul 01, 2023 |
| Contact name |
Joseph Mauro Calabrese |
| E-mail(s) |
mauro_calabrese@med.unc.edu
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| Organization name |
UNC Chapel Hill
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| Department |
Pharmacology
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| Lab |
Mauro Calabrese
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| Street address |
120 Mason Farm Rd
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27514 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE217239 |
Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn [RNA-Seq] |
| GSE217262 |
Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn |
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| Relations |
| BioSample |
SAMN31605425 |
| SRA |
SRX18169320 |
| 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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