|
| Status |
Public on Feb 05, 2021 |
| Title |
HeLa_HS_siZNF451_Slam-seq_REP2 |
| Sample type |
SRA |
| |
|
| Source name |
HeLa
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
treatment: siZNF451
condition: Heat-Shock + Recovery
|
| Treatment protocol |
Heat-shock and labeling was carried out 24 hours after the change of media (48 hours after transfection and/or knockdown) in the following manner: Cells were subject to heat shock at 43 degrees Celsius for 30 minutes, with labeling beginning 15 minutes into heat-shock with 200uM 4SU (Caymen Chemical 16373), after a total of thirty minutes of heat-shock, cells were returned to 37 degrees Celsius for 90 minutes of recovery. The total labeling time was 105 minutes.
|
| Growth protocol |
HeLa cells were plated, and after 24 hours for experiment (1) NELF-A Wildtype was knocked down using Lipofectamine RNAiMAX with concurrent transfection with either NELF-A WT or NELF-A ΔIDR using Lipofectamine 3000, for experiment (2) scr siRNA and siZNF451 was used with Lipofectamine RNAiMAX all per manufacturers instructions. Media was changed 24 hours after transfection and/or knockdown.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol per manufactures protocol, with addition of DTT, and protection from light during the procedure to prevent S-S cross-binding.
5 ug of RNA was used for SLAMseq modifications, as described in Herzog et. al. 2017. Briefly, Iodoacetamide (Sigma I1149) was conjugated to 4SU in a 50mM pH 8.0 phosphate buffer in DMSO/Water (1:1), the reaction was quenched with DTT and RNA was reprecipitated using Ethanol and NaOAc. Libraries were prepared using Quantseq 3’ mRNA-Seq Library Prep Kit (Lexogen).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: Slam-seq
3'UTR annotations were taken from Gencode annotation release 31 and merged on a gene level. Adapters and polyA stretches were trimmed from raw reads using fastp. Trimmed reads were further processed with SlamDunk v0.3.4. "Slamdunk all" command was executed with default parameters except '-5 12 -n 100 -t 20 -m -rl 100 --skip-sam'. Differential gene expression was done with DESeq2 using raw read counts with at least 2 T>C conversions. Size factors were calculated based on corresponding total read counts for global normalization.
Genome_build: hg38
Supplementary_files_format_and_content: slamdunk_mapped_filtered_tcount.tsv
|
| |
|
| Submission date |
Feb 11, 2020 |
| Last update date |
Feb 06, 2021 |
| Contact name |
Barbara Hummel |
| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
|
| Street address |
Stübeweg 51
|
| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE140053 |
Stress-induced nuclear condensation of NELF drives global transcriptional downregulation |
|
| Relations |
| BioSample |
SAMN14083398 |
| SRA |
SRX7708038 |
