|
| Status |
Public on Apr 01, 2020 |
| Title |
TC-797_DMSO_replicate4 (TT-seq) |
| Sample type |
SRA |
| |
|
| Source name |
TC-797 cultured cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: NUT carcinoma
cell line: TC-797
spike-in: Drosophila melanogaster Kc167 cell 4-thiouridine-labelled total RNA
|
| Treatment protocol |
MZ1 (Cayman Chemical #21622) was solubilized in DMSO. Triptolide (TPL; Cayman Chemical #11973) was solubilized in DMSO. TC-797 cells were treated with 100 nM MZ1 (0.01% final DMSO) or 500 nM triptolide (0.01% final DMSO) for 4 hours. For washout experiments, TC-797 cells were treated with 100 nM MZ1 (0.01% final DMSO) for 4 hours, washed three times with PBS, and then returned to normal culture media (as described above) for 24 hours.
|
| Growth protocol |
TC-797 cells were a kind gift from Jeffrey A. Toretsky (Georgetown University, Washington, DC). Cells were grown in DMEM with 4.5 g/L glucose and sodium pyruvate without L-glutamine (Corning #15013CV) + 10% Fetal Bovine Serum (Atlanta Biologicals #S10350) + 1% GlutaMAX (Gibco #35050061) + 1% Penicillin-Streptomycin (Gibco #15140122) at 37° C with 5% CO2. Kc167 cells were obtained from the Drosophila Genomics Resource Center (#1; Bloomington, IN). Cells were grown in CCM3 Media (HyClone #SH30062.02) at 25° C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed with TRIzol (Thermo Fisher #15596018) and total RNA was isolated essentially following the manufacturer’s instructions. Based on the yield of total RNA the amount of total RNA per 10 million cells was determined and 400 ng of 4-thiouridine labelled total Drosophila Kc167 cell RNA was added/spiked-in to an aliquot of total RNA corresponding to 10 million TC-797 cells. RNA was fragmented using a NEBNext RNA Fragmentation Module and then labelled with MTSEA-biotin-XX. Labelled RNA was enriched using streptavidin Dynabeads.
rRNA was depleted from the sample using a NEBNext rRNA Depletion Kit (New England Biolabs #E6310). A DNA library (from reverse transcribed RNA) was prepared for high-throughput sequencing following chapter 5 in the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs #E7760).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Reads were trimmed for adaptor sequences using trimmomatic v0.36.
Reads were aligned to concatenated hg38/Genome Reference Consortium Human Reference 38 Patch 15 (GRCh38.p15) and dm6/BDGP Release 6 + ISO1 MT assemblies of the human and Drosophila melanogaster genomes, respectively, using STAR v2.5.2b.
Duplicate and multimapper reads were marked with STAR with option --bamRemoveDuplicatesType UniqueIdentical and then removed with samtools v1.3.1 with option -F 1024.
STAR was used to compute signal tracks with options --outWigType bedGraph --outWigStrand Stranded --outWigNorm RPM.
TT-seq profiles were calibrated using spike-in normalization with D. melanogaster as the calibration genome, as previously described (Lugowski et al., 2018).
Reads overlapping genes were counted using htseq-count v0.9.1 (Anders et al., 2015) with parameters -f bam -r pos -s reverse -t gene -i gene_id. The gene set used consisted of protein-coding genes and lincRNAs from human GENCODE v24 (gtf column “transcript_type” either “protein_coding” or “lincRNA”) concatenated with those from FlyBase release 6.21.
Expression levels (fpkm) and differentially expressed genes were determined using DESeq2 v1.18.1 (Love et al., 2014), excluding genes with less than 10 counts in all samples, using size factors determined from the D. melanogaster gene counts, and calling significance at a Benjamini-Hochberg adjusted p-value < 0.01.
Genome_build: GRCh38.p15 concatenated with dm6/BDGP Release 6 + ISO1 MT
Supplementary_files_format_and_content: *_plus.bw file of plus-strand TT-seq track in bigWig format.
Supplementary_files_format_and_content: *_minus.bw file of minus-strand TT-seq track in bigWig format.
Supplementary_files_format_and_content: *_FPKM.bed file of per gene expression levels as FPKM values.
Supplementary_files_format_and_content: *_DESeq2.csv file of differential gene expression analysis between indicated conditions.
|
| |
|
| Submission date |
Oct 22, 2019 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Kyle Eagen |
| E-mail(s) |
kyle.eagen@bcm.edu
|
| Organization name |
Baylor College of Medicine
|
| Department |
Department of Molecular and Cellular Biology
|
| Lab |
Eagen Lab
|
| Street address |
1 Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL20795 |
| Series (2) |
| GSE133164 |
Transient Transcriptome Sequencing (TT-seq) of Nascent Gene Expression upon BRD4-NUT Protein Degradation/Recovery and RNA Polymerase II Inhibition |
| GSE133165 |
Chromatin Hyperacetylation Impacts Chromosome Folding by Forming a Nuclear Subcompartment |
|
| Relations |
| BioSample |
SAMN13087209 |
| SRA |
SRX7035649 |
