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| Status |
Public on Apr 13, 2017 |
| Title |
K562 dCas9-KRAB, Lenti-sgRNA separate infection, beta-globin pilot, batch 4 |
| Sample type |
SRA |
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| Source name |
K562 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
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| 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. Immediately prior to library construction, the separately infected cells were combined to a single tube.
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| Growth protocol |
K562 were cultured in IMDM plus 10% FBS and pen/strep at 37C and 5% CO2.
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
sgrnas and barcodes: listed in supplementary document
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| 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
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| Submission date |
May 23, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gary Chung Hon |
| Organization name |
UT Southwestern
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| Department |
OB/GYN
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE81748 |
Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of beta-globin locus (separate infection) |
| GSE81884 |
Multiplexed engineering and analysis of endogenous enhancer activity in single cells |
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| Relations |
| BioSample |
SAMN05176102 |
| SRA |
SRX1799670 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2175045_K562_dCas9-KRAB_Lenti-sgRNA_separate_infection_beta-globin_pilot_batch_4.cell_barcode_to_sgRNA_barcode.txt.gz |
1.0 Kb |
(ftp)(http) |
TXT |
| GSM2175045_K562_dCas9-KRAB_Lenti-sgRNA_separate_infection_beta-globin_pilot_batch_4.counts.txt.gz |
3.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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