|
| Status |
Public on Jan 06, 2020 |
| Title |
RNA-seq_CTCF Y226A, F228A_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
CTCF Y226A, F228A
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
genotype: CTCF Y226A, F228A
|
| Treatment protocol |
gRNA targeting exon 1 of CTCF were designed and annealed into pX330 (primer: 5’- CGATTTTGAGGAAGAACAGC-3’). To modify the targeted locus we co-transfected a 120 basepair repair oligo containing the desired mutation and a silent mutation. For oligo sequence, see supplementary file on the GSE126637 SuperSeries record. pBabePuro was co-transfected in a 10:1 ratio to the pX330. Transfected clones were selected using 2 µg/µl puromycin for 2 days. gDNA of clones was isolated and send for Sanger sequencing to check for the desired mutation.
|
| Growth protocol |
HAP1 cells (Carette et al., 2011) were cultured in IMDM (Invitrogen) supplemented with 10% FCS (Clontech), 1% Penicillin-Streptomycin (Invitrogen) and 0.5% Ultraglutamin (Lonza).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen).
Strand-specific libraries were generated using the TruSeq PolyA Stranded mRNA sample preparation kit (iIlumina). In brief, polyadenylated RNA was purified using oligo-dT beads. Following purification, the RNA was fragmented, random-primed and reserve transcribed using SuperScript II Reverse Transcriptase (Invitrogen). The generated cDNA was 3’ end-adenylated and ligated to Illumina Paired-end sequencing adapters and amplified by PCR using HiSeq SR Cluster Kit v4 cBot (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
CTCF_103_01
5235_4_CTCF
|
| Data processing |
Reads were mapped to hg19 using TopHat 2.1.1 (Kim et al., 2013) and HTSeq-count (Anders et al., 2015) was used to generate transcript-counts using GENCODE release 19 as reference.
DESeq2 was used to infer differentially expressed genes with an adjusted p-value threshold of 0.05.
Genome_build: hg19
Supplementary_files_format_and_content: Count table of reads per transcript for all samples and DESeq2 results of differential expression for the CTCF mutant compared to WT.
|
| |
|
| Submission date |
Feb 15, 2019 |
| Last update date |
Jan 06, 2020 |
| Contact name |
Angela Sedeno Cacciatore |
| E-mail(s) |
a.sedeno.cacciatore@nki.nl, m.v.ruiten@nki.nl
|
| Organization name |
Netherlands Cancer Institute
|
| Department |
Gene regulation
|
| Lab |
Rowland lab
|
| Street address |
Plesmanlaan 121
|
| City |
Amsterdam |
| State/province |
Noord-Holland |
| ZIP/Postal code |
1066 CX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE126635 |
The structural basis for cohesin/CTCF anchored loops [RNA-seq] |
| GSE126637 |
The structural basis for cohesin/CTCF anchored loops |
|
| Relations |
| BioSample |
SAMN10953883 |
| SRA |
SRX5383061 |
