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Sample GSM2175054 Query DataSets for GSM2175054
Status Public on Apr 13, 2017
Title K562 dCas9-KRAB, WT vs TNFA treatment, biorep2, batch 1
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. Titrated virus aliquot(s) were thawed to room temperature and one virus was added to each well. 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. One batch of cells were treated with TNF-alpha for 1 hour. Immediately prior to library construction, the TNF-alpha treated cells (labeled with sgRNA64) and mock treated cells (labeled with sgRNA63) were combined to a single tube in PBS/BSA.
Growth protocol K562 were cultured in IMDM plus 10% FBS and pen/strep at 37C and 5% CO2.
Extracted molecule total RNA
Extraction protocol Single cell RNA sequencing was performed using the Drop-Seq method. Briefly, single cells, polyA-containing barcoded beads (ChemGenes), and droplet generation oil (Bio-Rad) were each flowed into individual input ports on a microfluidic device (NanoShift). Cells in droplets were lysed and hybridized to beads. Beads were isolated and subjected to reverse transcription (Thermo Fisher) and exonuclease I (NEB) treatment, followed by PCR of full length transcripts (Kapa). Final libraries were created by Tn5-mediated tagmentation of cDNA, and amplified by PCR (Kapa) and purified by SPRI beads.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description sgrnas and barcodes: listed in supplementary document
Data processing Basecalls performed by Picard ExtractIlluminaBarcodes v1.117.
Drop-Seq data were mapped using Drop-Seq Tools to the human genome augmented with a sequence representing the sgRNA-containing plasmid with a fully degenerate 12-bp barcode sequence. Uniquely mapping reads with mapping quality ≥ 10 were subjected to PCR duplicate removal by examining the unique molecular identifiers (UMI) for each read. Reads were then partitioned to individual cells based on cell barcodes.
Read counts were tabulated with featureCounts v1.5.0.
Genome build: hg19
Supplementary_files_format_and_content: read counts for all genes
Submission date May 23, 2016
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)
GSE81749 Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq TNF-alpha treatment versus mock
GSE81884 Multiplexed engineering and analysis of endogenous enhancer activity in single cells
BioSample SAMN05176111
SRA SRX1799679

Supplementary file Size Download File type/resource
GSM2175054_K562_dCas9-KRAB_WT_vs_TNFA_treatment_biorep2_batch_1.cell_barcode_to_sgRNA_barcode.txt.gz 691 b (ftp)(http) TXT
GSM2175054_K562_dCas9-KRAB_WT_vs_TNFA_treatment_biorep2_batch_1.counts.txt.gz 3.6 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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