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| Status |
Public on Jul 15, 2021 |
| Title |
MV4-11_DMSO_RNA-seq_Rep1 |
| Sample type |
SRA |
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| Source name |
Cell culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MV4;11
treatment: DMSO
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| Treatment protocol |
Per growth protocol and treatment condition.
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| Growth protocol |
Exponentially growing MV4;11 cells were grown in 150 mm2 tissue culture-treated plates (Corning cat # 0877224) in 30 ml media ± 10 nM pinometostat for 4 or 7 days. Every 2 days cells were spun down at 500 x g 5 min. then resuspended in media ± 10 nM pinometostat. Cells were harvested on day 4 or 7.
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| Extracted molecule |
total RNA |
| Extraction protocol |
After 7 days of treatment, 1 x 107 cells were spun down at 500 x g 5 min. then cells were resuspended in 1 ml Trizol reagent (Life Technologies cat# 15596018), incubated 5 min. at RT, then 200 μl chloroform was added and samples were shaken rigorously for 15 sec. then incubated 3 min. at RT and spun down 12,000 x g 15 min. at 4 °C. The aqueous layer (~ 500 μl) was removed and mixed with 500 μl EtOH and added to a Zymo Research RNA Clean and Concentrator column (cat # 11-353B) and then spun at 12,000 x g for 1 min.. 100 μl DNase I (1:10 in buffered dH20) (Thermo Fisher Scientific cat # en0521) was added to the column and then it was spun at 500 x g 5 min., incubated 15 min. at RT and then spun 12,000 x g for 30 sec. and the flow through was discarded. After each of the following were added to the column, it was spun down 12,000 x g for 30 sec. and the flow through was discarded. Combined 200 μl RNA binding buffer with 300 μl EtOH and then spun 12,000 x g for 30 sec. and the flow through was discarded. After each of the following were added to the column, it was spun down 12,000 x g for 30 sec. and the flow through was discarded: 400 μl RNA prep buffer; 700 μl RNA wash buffer; and 400 μl RNA wash buffer. RNA was eluted from column with 30 μl RNase-free dH2O. Added RNA standards to 2 μg of each RNA sample- Add the equivalent of 10 copies/cell yeast RAD51; 30 copies/cell RNL2; 200 copies/cell E coli MBP; and 2000 copies/cell yeast SUMO to each sample then proceeded with rRNA removal Ribo Zero Gold kit (Illumina cat # MRZ11124C) according to manufacturer’s protocol. Add sample to Zymo RNA clean and concentrator columns and follow manufacturer's protocol. Elute in 7 μl RNase-free dH2O.
Libraries were prepared using the NEBNext Ultra Directional RNA Library prep kit (NEB cat # E7420S) with 5 μl of purified sample. Primer indices 4, 6 and 12 were used for DMSO-treated samples and 2, 7 and 9 for pinometostat-treated samples. Samples were amplified with 15 cycles of PCR. Libraries were then sequenced on the Illumina NextSEQ500 for paired-end sequencing, combining treated samples and untreated samples in separate lanes.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
For ChIP-seq analyses, analysis of histone methylation density (HMD) was carried out using the scripts and workflow from (Grzybowski et al. Nat Protoc. 2019;14(12)).
For RNA-seq analyses, reads were aligned to the index using HISAT2 (v 2.0.1) (Pertea et al., 2016). Cufflinks was used for differential gene expression analysis between the three DMSO-treated samples vs. the 10 nM pinometostat-treated samples.
Genome_build: hg38
Supplementary_files_format_and_content: BED files represent read locations genome-wide
Supplementary_files_format_and_content: BEDGRAPH files represent basepair depth or normalized depth
Supplementary_files_format_and_content: BIGWIG files represent basepair depth or normalized depth for genome browsers
Supplementary_files_format_and_content: FPKM_TRACKING files represent gene abundances for transcripts in RNA-seq experiment
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| Submission date |
Dec 01, 2020 |
| Last update date |
Jul 15, 2021 |
| Contact name |
Rohan Nishant Shah |
| E-mail(s) |
rohanshah@uchicago.edu
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| Organization name |
University of Chicago
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| Street address |
CLSC 856, 920 E. 58th Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE162441 |
MLL-fusion-mediated activation of FLT3-ITD lesions promotes leukemogenesis |
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| Relations |
| BioSample |
SAMN16970031 |
| SRA |
SRX9614746 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4952087_AR14-4-I-4.genes.fpkm_tracking.gz |
1.2 Mb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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