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Sample GSM3610141 Query DataSets for GSM3610141
Status Public on Jan 06, 2020
Title RNA-seq_WT_rep3
Sample type SRA
 
Source name WT
Organism Homo sapiens
Characteristics cell line: HAP1
genotype: Wild-type
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 WT_03
5235_3_WT
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 SAMN10953884
SRA SRX5383060

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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