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| Status |
Public on Jul 01, 2023 |
| Title |
E14 ESC Dox Airn-gRNA TRE-dCas9-VP160 H3K27me3 |
| Sample type |
SRA |
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| Source name |
E14 embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 embryonic stem cells
passage number: 30-35
genotype: Airn Highly-Expressing ESCs
chip antibody: Tri-Methyl-Histone H3 (Lys27) (C36B11) (H3K27me3) (Cell Signaling, cat #: 9733)
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| Treatment protocol |
Prior to crosslinking for ChIP, ESCs were induced with 1ug/mL doxycycline for 4 days and TSCs were passaged once off of irMEFs. For all ChIP experiments, except those for PRC components, adhered cells were crosslinked with 0.6% formaldehyde (Fisher Scientific, cat #: BP531-500) in RPMI media with 10% FBS for 10 minutes at room temperature, then quenched with 125mM glycine for 5 minutes at room temperature. Crosslinked cells were then washed twice with ice-cold PBS and scraped with ice-cold PBS with 0.05% Tween (Fisher Scientific, cat #: EW-88065-31) and PIC (Sigma Aldrich, cat #: P8340). The cells were then spun at 1,200 x g at 4°C to remove PBS, followed by resuspension in ice-cold PBS with PIC and divided into 10-million cell aliquots. For PRC component ChIPs, adhered C/B TSCs were crosslinked in PBS with 2mM DSG (disuccinimidyl glutarate; Thermo Scientific, cat #: 20593) for 45 minutes at room temperature and then in PBS with 1% formaldehyde (Thermo Scientific, cat #: 28906) for 15 minutes at room temperature. Crosslinking was quenched with 200mM glycine for 5 minutes at room temperature. Cells were then washed, scraped, and aliquoted as above.
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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. Mouse E14 ESCs were cultured on gelatin-coated plates in ESC media (DMEM high glucose and sodium pyruvate [Gibco, cat #: 11995073], 15% qualified FBS, 0.1mM MEM non-essential AA [Gibco, cat #: 11140050], 0.1mM penicillin-streptomycin, 2mM L-glutamine, 100M beta-mercaptoethanol, 1:500 LIF [ESC media conditioned with Lif-1Cα/COS cells]) at 37°C in a humidified incubator with 5% CO2. At passage, ESCs were trypsinized with 0.125% Trypsin-EDTA in PBS solution for ~5 minutes at room temperature and dislodged from the plate at single-cell suspension. ESCs were passaged every other day and provided fresh media daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For all ChIP DNA elution steps, washed beads were resuspended in Elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) and placed on a 65°C heat block for 17 minutes with frequent vortexing. RAD21 ChIP DNA was eluted for 1 hour under the same conditions. ChIP DNA was then reverse crosslinked in 0.5% SDS and 100mM NaCl overnight at 65°C, followed by a 1-hour RNaseA (3uL; Thermo Scientific, cat #: EN0531) treatment at 37°C and a 2.5-hour Proteinase K (10uL; Invitrogen, cat #: 25530015) treatment at 56°C. DNA was then extracted with 1 volume of phenol:chloroform:isoamyl alcohol (Sigma-Aldrich, cat #: P3803) and precipitated with 2 volumes 100% ethanol, 1/10 volume 3M sodium-acetate pH 5.4, and 1/1000 volume linear acrylamide (Invitrogen, cat #: AM9520) overnight at -20°C. Precipitated DNA was then extracted with a 30-minute max speed spin at 4°C, washed once with 80% ethanol, and resuspended in TE.
ChIP-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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
ChIP-Seq reads were aligned using bowtie2 with default parameters (Langmead and Salzberg 2012).
For all ChIP-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.
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| Submission date |
Dec 07, 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) |
| GSE217262 |
Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn |
| GSE220464 |
Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn [ChIP-Seq] |
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| Relations |
| BioSample |
SAMN32097907 |
| SRA |
SRX18532504 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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