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| Status |
Public on Mar 26, 2024 |
| Title |
CR137_LV3599_Control_midXL_MORC2 |
| Sample type |
SRA |
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| Source name |
Sai2
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Sai2
cell type: human neuroepithelial-like stem cells
cutandrun antibody: MORC2
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| Treatment protocol |
To silence the transcription of MORC2 and TASOR, we used the catalytically inactive Cas9 (deadCas9) fused to the transcriptional repressor KRAB (Johansson et al 2022). Single guide sequences were designed to recognize DNA regions just down-stream of the transcription start site (TSS) according to the GPP Portal (Broad Institute). The following lentiviral backbone was used: pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP. Lentiviruses were produced as described below yielding titers of 108-109 TU/ml, which was determined using qRT–PCR. Control virus with a gRNA sequence not present in the human genome (LacZ) was also produced and used in all experiments. GFP cells were FACS isolated (FACSAria, BD sciences) on day 10 at 10°C (reanalysis showed > 97% purity) and pelleted at 400 g for 5 min, snap frozen on dry ice and stored at −80°C until RNA isolation.
To do the DNMT1 knock out, we used a CRISPR cut approach. LV.gRNA.CAS9-GFP vectors were used to target DNMT1 (Liao et al 2015) or LacZ (control) as described in (Jönsson et al 2019). Lentiviral vectors were produced as described previously and had a titer of 108-109 TU/ml which was determined using qRT-PCR. hNSCs were transduced with a MOI of 10–15 and allowed to expand 10 days and were FACS sorted as described previously.
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| Growth protocol |
An embryo-derived human neural epithelial-like stem cell (hNSC) line, Sai2, was used (Tailor et al 2013). The hNSCs were cultured according to standard protocol (Falk et al 2012). Briefly, the cells were kept in DMEM/F12 (Thermo Fisher Scientific) supplemented with Glutamine (2mM, Sigma), Penicillin/Streptomycin (1x, Gibco), N2 (1x, Thermo Fisher Scientific), B27 (0.05x, Thermo Fisher Scientific), EGF and FGF2 (both 10ng/ml, Thermo Fisher Scientific). 10 mM Y27632 Rock inhibitor (Miltenyi) was also used. Cells were grown on Nunc multidishes or T25 flasks pre-coated with Poly L-Ornithine (15μg/ml, Sigma) and Laminin (2μg/ml, Sigma). Cells were passaged 1:3 every 2-3 days using TryplLETM Express enzyme (GIBCO) and Trypsin Inhibitor (GIBCO).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a E220 focused-ultrasonicator (Covaris).
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired-end reads (2x75 for CR136&CR137 and 2x150 for the rest of the sequencing runs) were aligned to the hg38 genome using bowtie2 v2.3.4.2 (--local–very-sensitive-local --no-unal –no-mixed –no-discordant –phred33 -I 10 -X 700)
SAM files were converted to bam files with SAMtools v1.9. The BAM files were filtered according to alignment quality (MAPQ>10) using samtools (samtools view -bq 10 {BAM} > {FILTERED_BAM}).
Normalized bigwig coverage tracks were made with bamCoverage v2.4.3, with RPKM normalization.
GRCh38
Assembly: Bigwigs of filtered alignments for genome browser visualization, normalized with RPKM
Library strategy: CUT&RUN-Seq
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| Submission date |
Aug 08, 2023 |
| Last update date |
Mar 27, 2024 |
| Contact name |
Ninoslav Pandiloski |
| E-mail(s) |
[email protected]
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| Organization name |
Lund University
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| Street address |
Sölvegatan 17
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| City |
Lund |
| ZIP/Postal code |
223 62 |
| Country |
Sweden |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE240384 |
DNA methylation status determines the sensitivity of repeats to restriction by the HUSH-MORC2 complex [CUT&RUN] |
| GSE242143 |
DNA methylation status determines the sensitivity of repeats to restriction by the HUSH-MORC2 complex |
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| Relations |
| BioSample |
SAMN36892547 |
| SRA |
SRX21301530 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7697283_CR137_LV3599_Control_midXL_MORC2_mapq10.bw |
118.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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