|
| Status |
Public on Jul 16, 2021 |
| Title |
Control_Polysome4 |
| Sample type |
SRA |
| |
|
| Source name |
human neuroblastoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: SH-SY5Y
treatment: Control (DMSO)
|
| Treatment protocol |
Neuronal differentiation was performed using retinoic acid (RA) at passage 4. All trans Retinoic Acid (RA, Sigma) was added to cells 24h after plating, at final concentration of 30μM.
|
| Growth protocol |
Human neuroblastoma SH-SY5Y cells, were cultured in Dulbecco’s Modified Eagle Medium (DMEM 4.5g/L Glucose with L-Glutamine) supplemented with 1% (v/v) Penicillin/Streptomycin and 10% Fetal Bovine Serum (FBS) at 37oC, 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA purification from RNaseI footprinted samples was performed by Trizol RNA extraction. RNA purification from polysome fractions was performed by isopropanol precipitation, followed by TURBO DNase treatment, acidic phenol/chloroform RNA purification and ethanol precipitation at -80oC overnight. RNA concentration was determined by Nano-drop 2000 software. Two rounds of polyA selection from polysome fractions were performed using oligo (dT) Dynabeads PolyA RNA was fragmented by alkaline hydrolysis. 28–34 nt ribosome footprints and 50–80 nt mRNA fragments were gel purified in 10% (w/v) polyacrylamide-TBE-Urea gel. Ribosome footprints were subjected to rRNA depletion (Illumina RiboZero rRNA removal kit).
5’ stranded libraries were constructed using NEB Next Multiplex Small RNA Library Prep. Resulting cDNA was PCR amplified and gel purified prior to sequencing. Libraries were subjected to 75bp single end RNA Sequsing NextSeq 500 Illumina sequencer,High OutputKit v2.5 (75 Cycles)(Next Generation Sequencing Facility, Faculty of Medicine, University of Leeds).
RNA-seq, Ribo-seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
polysome-associated RNA
Control_FPP4_translated_ORFs_filtered_sorted.bed
|
| Data processing |
Adaptor sequences were trimmed using Cutadapt (v.2.8) with minimum read length of 25bp, and untrimmed outputs retained for RNA-seq reads.
Low-quality reads (score <20 for 10% or more of read) were discarded using FASTQ Quality Filter, FASTX-Toolkit (v.0.0.14).
Reads aligning to rRNA and tRNA sequences were discarded using Bowtie 2 (v2.3.5) and one base was removed from the 3' end of reads.
Unaligned Ribo-seq reads and RNA-seq reads were aligned to the Human reference genome (Gencode v30) using the split-aware aligner STAR (v2.6.1b). The STAR genome index was built with an sjdbOverhang of 73.
Alignments flagged as secondary alignment were filtered out with samtools (v1.9), ensuring 1 genomic position per aligned read. "samtools view -b -F 0X100"
RiboTaper was applied to these data to find ORFs and quantify the p-site positions per ORF (reported in score column of processed data bed file).
Genome_build: GRCh38
Supplementary_files_format_and_content: bed file of open reading frames determined by RiboTaper pipeline.
|
| |
|
| Submission date |
Feb 04, 2021 |
| Last update date |
Jul 16, 2021 |
| Contact name |
Julie Aspden |
| Organization name |
University of Leeds
|
| Department |
FBS
|
| Street address |
Woodhouse
|
| City |
Leeds |
| ZIP/Postal code |
LS2 9JT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE166214 |
Cytoplasmic long non-coding RNAs are differentially regulated and translated during human neuronal differentiation |
|
| Relations |
| BioSample |
SAMN17799940 |
| SRA |
SRX10031271 |
