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| Status |
Public on Jul 01, 2023 |
| Title |
E14 ESC Dox Airn-gRNA TRE-dCas9-VP160 Airn CHART |
| 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: Airn Highly-Expressing ESC
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| Treatment protocol |
CHART was performed as in the detailed protocol from (Davis and West 2015). TSCs were passaged once off of irMEFs as above. Airn Highly-Expressing TSCs from (Schertzer et al. 2019a) and ESCs were induced with 1g/mL doxycycline 4 days prior to crosslinking. Adhered TSCs and ESCs were crosslinked with 1% formaldehyde (Fisher Scientific, cat #: BP531-500) in PBS solution for 10 minutes at room temperature. On ice, cells were washed twice with ice-cold PBS and twice with ice-cold PBS + 0.05% Tween before being scraped, spun at 1,000 x g for 5 minutes at 4°C, and divided into 25-million cell aliquots.
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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], 100M -mercaptoethanol [Sigma-Aldrich, cat #: 63689]) supplemented with 25ng/mL FGF4 (Gibco, cat #: PHG0154) and 1g/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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CHART-Seq libraries were prepared with NEBNext End Repair Module (NEB, cat #: E6050S), A-tailing by Klenow Fragment (3’--5’ exo-; NEB, cat #: M0212S), and TruSeq 6-bp index adaptor ligation by Quick ligase (NEB, cat #: M2200S), and NEBNext High-Fidelity 2X PCR Master Mix (NEB, cat #: M0541S). All DNA clean-up steps were performed with AMPure XP beads (Beckman Coulter, cat #: A63880). Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit (Illumina, cat #: 20024906) on a NextSeq 500 System.
Capture Hybridization Analysis of RNA Target DNA sequencing (CHART-Seq)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
CHART-Seq reads were aligned using bowtie2 with default parameters (Langmead and Salzberg 2012).
For all CHART-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).
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). Where indicated, the WIG file includes reads pooled from the two or more biological replicates.
Library strategy: CHART-Seq
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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) |
| GSE217261 |
Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn [CHART-Seq] |
| GSE217262 |
Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn |
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| Relations |
| BioSample |
SAMN31605351 |
| SRA |
SRX18169262 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6710222_e14_dox_dcas9-vp160-airn_airn-chart.wig.gz |
86.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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