|
| Status |
Public on Mar 09, 2022 |
| Title |
LiverB_WT_ONT |
| Sample type |
SRA |
| |
|
| Source name |
liver tissue from young adult wild-type FVB mice, circAID-p-seq/ONT
|
| Organism |
Mus musculus |
| Characteristics |
tissue: liver tissue
replicate: #2
rna fraction: Ribo-seq
extract_protocol: circAID-p-seq
sequencing platform: Oxford nanopore technology
treatment: untreated
|
| Treatment protocol |
HEK293T cells were treated with or without harringtonine (2 µg/mL) for 3 min, followed by CHX (10 μg/mL) for 5 min at 37°C.
|
| Growth protocol |
Young adult wild-type FVB mice were obtained from breeding stocks at the University of Edinburgh. HEK293 (H. Sapiens, RRID: CVCL_0045) cells were seeded at 1.5x106 cells/dish and kept in culture until reaching 80% of confluence
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Oxford nanopore libraries for RPFs from HEK293T and mouse liver were prepared according to circAID-p-seq protocol. Briefly, upon 5′ phosphorylation of RPFs, library preparation was performed starting from 10 pmol of ADR12 used for ligation, followed by RNA self circularization and RT-RCA. The final libraries were loaded on a R9.4 flow cell and sequenced with a MinION sequencer.
HEK298T cell lysates were obtained using a hypotonic lysis buffer. A total of 1.7 a.u. (A260 nm) were digested using . RPFs were generated by treating 1.7a.u. of lysate with 12.7 U of RNase I at room temperature for 45 min (as described in Clamer et al., 2018). ). Liver tissues were dissected immediately following sacrifice and pulverized under liquid nitrogen using a pestle and a mortar and the lysates obtained according to Bernabò et al.2017. Lysates were treated with RNAse I and the 80S with RPFs were isolated using sucrose gradient separation. The RPFs were purified using acidic phenol/chloroform extraction followed by PAGE extraction.
Libraries from RPFs isolated from HEK293T cells were prepared using the SMARTer® smRNA-Seq Kit for Illumina and sequenced with 50 cycles single-read on an Illumina NovaSeq 6000 sequencer. Illumina libraries for RPFs from mouse liver tissue were prepared according to Ingolia et al., 2012 and were sequenced with 50 cycles single-read on an Illumina HiSeq2500 sequencer.
Oxford nanopore libraries for RPFs from HEK293T and mouse liver were prepared according to circAID-p-seq protocol. Briefly, upon 5′ phosphorylation of RPFs, library preparation was performed starting from 10 pmol of ADR12 used for ligation, followed by RNA self circularization and RT-RCA. The final libraries were loaded on a R9.4 flow cell and sequenced with a MinION sequencer.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Description |
ribosome profiling with circAID-p-seq from mouse liver tissue
|
| Data processing |
Library strategy: circAID-p-seq
circAID-p-seq basecalling : Guppy 3.6.1
circAID-p-seq consensus generation: CircAidMe pipeline
Ribo-seq data from all the library construction were aligned on GRCh38.p13 for HEK293T and GRCm38.p6 for mouse tissue using Botwie 2
Alignment files were processed with Samtools version 1.9 and analyzed with the RiboWaltz R package
Genome_build: Human: GRCh38.p13 - Mouse:GRCm38.p6
Supplementary_files_format_and_content: Excel files including a brief annotation and transcript-specific counts of ribosome protected fragments
|
| |
|
| Submission date |
May 20, 2021 |
| Last update date |
Mar 09, 2022 |
| Contact name |
Alessia Del Piano |
| E-mail(s) |
[email protected], [email protected], [email protected]
|
| Phone |
0461312018
|
| Organization name |
IMMAGINA BioTECHNOLOGY
|
| Street address |
Via Sommarive 18
|
| City |
Trento |
| State/province |
Trento |
| ZIP/Postal code |
38123 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24973 |
| Series (1) |
| GSE174754 |
Phospho-RNA sequencing with CircAID-p-seq |
|
| Relations |
| BioSample |
SAMN19285886 |
| SRA |
SRX10947894 |
