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| Status |
Public on Jan 25, 2019 |
| Title |
SD4-9 |
| Sample type |
SRA |
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| Source name |
K562 cells
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| Organism |
Homo sapiens |
| Characteristics |
transfection: plasmid mixtures of Cas9-puro and U6-sgRNAs encoding each member of sgRNAs
cell type: human erythromyeloblastoid leukemia cell
cell line: K562
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| Treatment protocol |
Cells were transfected with plasmid mixtures of Cas9-puro, U6-sgRNA encoding sgRNAs at a weight ratio of 1:1:1 using Neon (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. One day after transfection, puromycin (Cat no. A11138-03, Invitrogen, Carlsbad, CA) was added to the culture media at a final concentration of 2.5 µgml-1 for two days. Three days after transfection, cells were analyzed.
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| Growth protocol |
K562, a human erythromyeloblastoid leukemia cell line, was purchased from American Type Culture Collection (Manassas, VA) and cultured in Roswell Park Memorial Institute medium (RPMI; Invitrogen, Carlsbad, CA) supplemented with 100 units ml-1 penicillin, 100 µg ml-1 streptomycin, and 10% fetal bovine serum.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Libraries were prepared according to illumina's instruction accompanying the Truseq stranded mRNA prep kit. Briefly, The polyA containing mRNA molecules were purified using poly-T oligo attached magnetic beads and then fragmented into small pieces. The cleaved RNA fragments were primed into random hexamers into first strand cDNA using reverse transcriptase and random primers. The RNA template removed and a replacement strand synthesized, incorporating dUTP in place of dTTP to generate ds cDNA. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were then purified and enriched with PCR to create the final cDNA library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
SD4-9
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| Data processing |
Basecalls performed using CASAVA version 1.8.2
The RNA-seq data for clone 10 was mapped to the human reference genome (hg19) using Bowtie version 1.0.0 (Trapnell et al., 2009) with unique-mapping parameter "-m 1".
To measure expression level, we estimated both reads per million reads (RPM) values on the GENCODE annotation version 19.
After whole genome sequencing (WGS) of the parental cells was performed, the reads were mapped using BWA version 0.7.10-r789, duplicates were marked with Picard version 1.96, realigned based on the presence of an insertion or deletion, and the quality scores recalibrated using GATK version 3.4.
The variants were called using GATK HaplotypeCaller version 3.4.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include transcript name, gene name, coordinates, RPKM values and RPM values
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| Submission date |
Jan 07, 2019 |
| Last update date |
Jan 25, 2019 |
| Contact name |
Hyeon Joo Lee |
| E-mail(s) |
hyeonlee17@gmail.com
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| Organization name |
Hanyang University
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| Department |
Department of Life Science
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| Lab |
BIG Lab.
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| Street address |
222 Wangsimni-ro, Seongdong-gu
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| City |
Seoul |
| State/province |
- Please choose - |
| ZIP/Postal code |
133-791 |
| Country |
South Korea |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE65830 |
En bloc and segmental deletions of human XIST reveal X chromosome inactivation-involving RNA elements |
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| Relations |
| BioSample |
SAMN10700169 |
| SRA |
SRX5213198 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3544624_K562_SD4_clone9_rpm.txt.gz |
901.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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