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| Status |
Public on Mar 26, 2024 |
| Title |
ONT_cDNAseq_LV3367_DNMT1_CRISPRcut |
| 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
genotype: DNMT1 CRISPRcut
treatment: CRISPR cut
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| Treatment protocol |
To silence the transcription of DNMT1 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.
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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 |
total RNA |
| Extraction protocol |
1.5 μg total RNA was isolated as above for each of the four samples tested (Control, MORC2-CRISPRi, TASOR-CRISPRi and DNMT1-KO hNPCs, 10 days post-transduction in each case). Ribosomal RNA was depleted using the RiboMinus Eukaryote System v2 (ThermoFisher). 5 ng of the resulting RNA was used to make barcoded cDNA libraries (2 barcodes per sample) using the cDNA-PCR kit SQK-PCB-111-24 (Oxford Nanopore Technologies), which incorporates a strand-switching step to enrich for full-length transcripts. Fifteen PCR cycles were used with 7 min extension time. Inspection of the final cDNA product by capillary electrophoresis confirmed a peak at around 2-2.5 kb, approximately the size of an average human mRNA. The libraries were sequenced with a FLO-PRO002 PromethION Flow Cell R9 Version on a PromethION (Oxford Nanopore Technologies) at SciLifeLab
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
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| Description |
ONT cDNA sequencing
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| Data processing |
Long read DNA sequencing data were aligned to the human genome (GRCh38) using minimap2 (-k15 -w5 -uf -x splice -O6,24).
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.
Assembly: GRCh38
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| Submission date |
Mar 26, 2024 |
| Last update date |
Mar 26, 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 |
GPL26167 |
| Series (2) |
| GSE242143 |
DNA methylation status determines the sensitivity of repeats to restriction by the HUSH-MORC2 complex |
| GSE262483 |
DNA methylation status determines the sensitivity of repeats to restriction by the HUSH-MORC2 complex [ONT cDNA sequencing] |
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| Relations |
| BioSample |
SAMN40620022 |
| SRA |
SRX24066010 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8170413_ONT_cDNAseq_LV3367_DNMT1_CRISPRcut.mapq10.bw |
5.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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