|
| Status |
Public on Jan 28, 2021 |
| Title |
hESC ZNF91 wt5 RNA seq rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
hESCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: H9 Human embryonic stem cells
genotype: ZNF91 wt
crispr clone: wt5
|
| Treatment protocol |
H9 hESCs were transfected with two gRNAs targeting ZNF91 cloned into pX330-SpCas9-HF1 and clonally expanded
|
| Growth protocol |
H9 human embryonic stem cells were grown on matrigel (Corning) coated dishes. They were cultured in hESC medium that was incubated with mouse embryonic fibroblasts (MEFs) for 24 hours. HESC medium consisted of: DMEM/F12 supplemented with 2mM L-glutamine (Invitrogen), 20% knockout serum replacement (Gibco), penicillin/streptomycin (Invitrogen), non-essential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Invitrogen). Medium was supplemented with basic fibroblast growth factor (sigma, 8ng/μl) and changed daily to secure pluripotency of hESCs. For maintenance of the culture, cells were grown in colonies and passaged manually by cutting the colonies with a needle
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol according to the manufacturer's protocol, followed by a DNAse treatment and RNA purification using the RNA clean and concentrator kit (Zymo research). Ribosomal RNA was depleted from total RNA with the rRNA depletion kit (NEB# E6310)
NEB Next Ultra Directional RNA Library Prep Kit (NEB #E7420) was used to prepare Samples for sequencing at an illumina Hiseq 4000 device.
RNA seq on a Hiseq 4000 device
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
RNAseq_ZNF91_hESC_DEseq2_ko9-13-19_vs_wt5-30_Basemean>10reads-in-1samp_SVAs.txt RNAseq_ZNF91_hESC_DEseq2_ko9-13-19_vs_wt5-30_genes.csv RNAseq_ZNF91_hESC_scaled_counts_wt5_wt30_ko9-13-19_genes.txt RNAseq_ZNF91_hESC_scaled_counts_wt5_wt30_ko9-13-19_repeats-all.txt RNAseq_ZNF91_hESC_scaled_counts_wt5_wt30_ko9-13-19_SVAs-all.txt
|
| Data processing |
Read quality assessment using FASTQC
Trimmomatic (Bolger et al. 2014) was used to clip adapter sequences and trim low quality reads.
Reads were aligned to the human genome (Hg19 version) using STAR (Dobin et al. 2013) with default settings except: outFilterMismatchNmax=2 and outFilterMultimapNmax=10, outWigType=bedGraph, outSAMtype=BAM SortedByCoordinate
Raw read counts of genes were determined with FeatureCounts (Liao et al. 2014) using Hg19 KnownGenes.GTF from UCSC for annotation (downloaded 12th September 2016). Only properly paired reads were counted (-B) and the library was reversely stranded (-s 2). Genes were summarized at metalevel (default), whereas TEs were summarized at the feature level (-f)
DEseq2 (Love et al. 2014) was used to normalize raw counts, perform principal component analysis and differential expression analysis of genes and repeats. For differential expression analysis of SVA elements Log2FC >3 was considered differentially expressed, because SVA expression levels were too low to determine adjusted p values adequately
Hg19
tab-delimited text files include scaled read counts for each Sample
Tab-delimited text/csv DEseq2 output files
|
| |
|
| Submission date |
Dec 02, 2020 |
| Last update date |
Jan 28, 2021 |
| Contact name |
Frank M.J. Jacobs |
| Organization name |
University of Amsterdam
|
| Department |
Sammerdam Institute for Life Sciences
|
| Lab |
Evolutionary Neurogenomics
|
| Street address |
Science Park 904
|
| City |
Amsterdam |
| ZIP/Postal code |
1098XH |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE162571 |
Genetic deletion of ZNF91 in human embryonic stem cells leads to ectopic activation of SVAs and collective upregulation of KRAB zinc finger gene clusters |
|
| Relations |
| BioSample |
SAMN16984611 |
| SRA |
SRX9621859 |
