|
| Status |
Public on Apr 08, 2016 |
| Title |
48h A771726 treated A375 cells replicate a |
| Sample type |
SRA |
| |
|
| Source name |
A375 cells, 25 μM A771726
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A375
cell type: malignant melanoma
assayed molecule: nascent RNA
|
| Treatment protocol |
One million A375 cells per condition were treated with DMSO, 25 μM leflunomide, or 25 μM A771726 in duplicate. Drugs used were leflunomide (Enzo Life Sciences) and A771726 (Enzo Life Sciences). After a 48 hr treatment, cells were washed, swelled, and lysed in lysis buffer with a reduced amount of IGEPAL detergent (composition: 10 mM Tris-Cl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 10% glycerol, 2 U/mL SUPERasin (Ambion), and 0.25% IGEPAL).
|
| Growth protocol |
Human A375 malignant melanoma cells (ATCC) are grown on standard tissue culture plates in filter sterilized DMEM (Life Technologies) with 10% heat-inactivated FBS (Atlanta Biologicals), 1X GlutaMAX (Life Technologies) and 1% Penicillin-Streptomycin (Life Technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After drug treatment, cells were frozen down, nuclear run-on reaction was performed, and nascent RNA was isolated.
TruSeq small RNA sample prep kit adapters and primers (Illumina) were used to make the samples suitable for multiplexing. The PCR reaction was run for 18 cycles. The indexed libraries were purified on a 6% native polyacrylamide gel, and were cut out between 150 and 300 bp. The concentration of the isolated libraries was estimated with a high-sensitivity DNA chip from Agilent according to manufacturer’s protocol and libraries were mixed in equal quantities and sequenced on Illumina Hi-Seq2000. Index sequences from multiplexed primers were used to identify treatment group and reads were demultiplexed using a perl script.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Gro-seq
|
| Data processing |
For each replicate of DMSO (control), Leflunomide and A771762 treated samples, reads were trimmed at the 3′ends to remove any linker sequence contamination.
Trimmed reads for each sample were aligned to the hg18 version of the human genome using Bowtie 0.12.9 with parameters -k 1 -m 10 -n 2 --best --strata.
RNA repeats were then filtered out from the alignments using bedtools.
Read density at the promoters and within the gene bodies were respectively calculated as the read density within 300 bp of the TSS, and the read density within the TSS +300 bp to the TTS +3 kb. Densities were normalized to the read density at the beta-Actin promoter for each sample and expressed as a fraction of millions of mapped reads per base pair (rpm/bp). The read densities were calculated on the transcripts which have a TR in the DMSO control condition initially low (TR< 7.5), representing 22442 over 47021 transcripts.
RNA Polymerase II Travelling ratios (TR) were calculated as the ratio of read densities at the promoters over the read densities within the gene bodies for each transcript in each sample. Average TRs were calculated from the TRs calculated in two experimental replicates.
Genome_build: hg18
Supplementary_files_format_and_content: Data table with the Traveling ratio calculated in each DMSO control, Leflunomide treated or A771726 treated conditions and a data table containing the read densities at the promoter and gene bodies.
|
| |
|
| Submission date |
May 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Leonard Zon |
| E-mail(s) |
zon@enders.tch.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Department |
Oncology/Hematology
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE57430 |
HEXIM1 is induced by DHODH inhibition to suppress melanoma [Gro-Seq] |
| GSE57432 |
HEXIM1 is induced by DHODH inhibition to suppress melanoma |
|
| Relations |
| BioSample |
SAMN02767785 |
| SRA |
SRX535449 |
