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Sample GSM2544707 Query DataSets for GSM2544707
Status Public on Apr 13, 2017
Title K562, dCas9-KRAB, infected with sgRNA64 (rep3); treated with TNFalpha for 1h
Sample type SRA
 
Source name K562 cells
Organism Homo sapiens
Characteristics cell line: K562
Treatment protocol HEK293T cells were cultured in DMEM with 10% FBS and pen/strep. For lentiviral packaging, 3X106 293T cells were seeded in a 6 cm dish one day before transfection. The indicated viral plasmid(s) were co-transfected with lentivirus packaging plasmids pMD2.G and psPAX2 (Addgene ID 12259 and 12260) with 4:2:3 ratio by using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's protocol. Twelve hours after transfection, media was changed to fresh DMEM with 10% FBS plus Pen/Strep. Seventy-two hours after transfection, virus-containing media was collected, passed through a 45 um filter, and aliquoted into 1.5ml tubes. Viruses were stored in -80°C before infection or titration. For virus infection, 2X105 K562 cells were seeded in 24 well plate per well with 500 µl complete medium plus 1µg/ml polybrene. Virus aliquot(s) were thawed to room temperature and added to the plates. The plate was then centrifuged at 1000xg at 36 °C and returned to the incubator. The following day, the media was changed to fresh complete medium with antibiotics to screen for infected cells. Cells were kept at ~30% confluence during antibiotic selection.
Growth protocol K562 were cultured in IMDM plus 10% FBS and pen/strep at 37C and 5% CO2.
Extracted molecule total RNA
Extraction protocol total RNA was extracted from cells using Trizol Reagent (Thermo), and were subjected to enrichment of polyA+ RNA using the NEBNext Poly(A) kit (NEB).
Strand-specific polyA+ RNA-Seq was performed using the LM-Seq method, as described previously. Briefly, PolyA+ RNA was fragmented at 85°C in SmartScribe first-strand buffer (CloneTech) supplemented with a 3’ sequencing adapter containing a random hexamer sequence. The fragmented RNA were subjected to first strand cDNA synthesis, followed by RNase A/H treatment. The remaining single-stranded DNA were ligated to a 5’ sequencing adapter. The libraries were then amplified by PCR using Illumina TruSeq-compatible primers and purified using SPRI purification beads.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description PZ309
sgRNA ID: sgRNA64
sgRNA barcode ID:
Data processing Basecalls performed bcl2fastq v2.17.1.14.
RNA-seq reads were aligned using the STAR aligner v2.4.2a with default parameters to the hg19 genome assembly augmented with a sequence representing the sgRNA-containing plasmid with a fully degenerate 12-bp barcode sequence.
Mapped reads were filtered for mapping quality >= 10, and PCR duplicates were removed with Picard v1.117.
Read counts were tabulated with featureCounts v1.5.0.
Genome build: hg19
Supplementary_files_format_and_content: read counts for all genes
 
Submission date Mar 20, 2017
Last update date May 15, 2019
Contact name Gary Chung Hon
Organization name UT Southwestern
Department OB/GYN
Street address 5323 Harry Hines Blvd.
City Dallas
State/province TX
ZIP/Postal code 75390
Country USA
 
Platform ID GPL18573
Series (2)
GSE81412 Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Bulk RNA-Seq validation
GSE81884 Multiplexed engineering and analysis of endogenous enhancer activity in single cells
Relations
BioSample SAMN06622063
SRA SRX2657966

Supplementary file Size Download File type/resource
GSM2544707_K562_dCas9-KRAB_infected_with_sgRNA64_rep3_treated_with_TNFalpha_for_1h.counts.txt.gz 3.1 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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