|
| Status |
Public on Apr 13, 2017 |
| Title |
K562 dCas9-KRAB, not infected |
| 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 3â 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 5â 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 |
sgrna barcode id: none
sgrna id: none
|
| 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.
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 |
May 13, 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) |
| 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 |
SAMN04998768 |
| SRA |
SRX1759554 |
