|
| Status |
Public on Mar 01, 2020 |
| Title |
HeLa_tRNA_3 |
| Sample type |
SRA |
| |
|
| Source name |
Human cervical adenocarcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
cell type: Human cervical adenocarcinoma cells
atcc number: CCL-2
passages: 16
|
| Growth protocol |
HeLa cells were grown in complete DMEM to 80% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Media was aspirated and Trizol was added immediately to culture dishes. Total RNA was isolated using Trizol manufacturer's instructions (ThermoFisher) followed by treatment with Turbo Dnase (ThermoFisher).
tRNA-sequencing: 1 micrograms of total RNA was ligated to annealed adapters under conditions as in (Shigematsu et al., 2017; PMID: 28108659) using 10-fold more RNA ligase. cDNA was synthesized using SuperScript IV Reverse Transcriptase (ThermoFisher 18090010) using manufacturer’s recommended conditions and gel purified with denaturing polyacrylamide gel electorphoresis. Gel purified cDNA was then ligated using CircLigase (Lucigen CL4111K) with manufacturer’s recommended conditions and libraries were amplified using Q5 DNA polymerase (New England Biolabs M0491) for 7-8 cycles then purified from a 2% agarose gel. Libraries were sequenced as single-end reads on an Illumina NextSeq 550.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
HeLa_tRNA_gene_summary_cyto_RPM.csv
HeLa_tRNA_anticodon_summary_RPM.csv
|
| Data processing |
Supplementary_files_format_and_content: HeLa_tRNA_gene_summary_cyto_RPM.csv - CSV, Reads per million (RPM) from HeLa tRNA-seq per unique tRNA gene sequence
Supplementary_files_format_and_content: HeLa_tRNA_anticodon_summary_RPM.csv - CSV, Reads per million (RPM) from HeLa tRNA-seq per anticodon group
Processing: cutadapt ver 1.12 was used twice in succession with the following parameters first to remove potential 5' adapter (-g ^ACTGGATACTGG) and then to remove the 3' CCA + adapter (-a CCAGTATCCAGTTGGAATT).
Mapping: bowtie2 ver 2.3.5 was used to map processed reads to a reference generated from high confidence tRNAs (unique sequences collapsed) defined in GtRNAdb (hg38) using the following parameters: --quiet --min-score L,1,0 --local -D 20 -R 3 -N 1 -L 10 -i S,1,0.5.
Downstream analyses: Mapped sam files were converted to bam files using samtools ver 1.9 (samtools view). Further analyses were carried out in Rstudio ver 1.1.463 (R ver 3.5.2 (2018-12-20) -- "Eggshell Igloo"). Read count tables over tRNA genes were generated by using featureCounts from the Rsubread package using the following parameters: strandSpecific = 1,useMetaFeatures=F,minMQS = 10.
Genome_build: hg38
|
| |
|
| Submission date |
Oct 02, 2019 |
| Last update date |
Mar 01, 2020 |
| Contact name |
Jeff Coller |
| E-mail(s) |
jmc71@case.edu
|
| Phone |
216-368-0299
|
| Organization name |
Case Western Reserve University
|
| Department |
Center for RNA Science and Therapeutics
|
| Street address |
10900 Euclid Ave
|
| City |
Cleveland |
| State/province |
OH |
| ZIP/Postal code |
44106 |
| Country |
USA |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE138292 |
Codon and amino acid content regulate mRNA stability in mammalian cells |
|
| Relations |
| BioSample |
SAMN12886787 |
| SRA |
SRX6935341 |
