|
| Status |
Public on Jan 07, 2023 |
| Title |
HEK 4SU PD 8 15 min SN |
| Sample type |
SRA |
| |
|
| Source name |
HEK Pulldown
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Flp-In T-REx 293 cell line
treatment: 1 mM 4-thiouridine 15 min
fraction: whole cell
pulldown reagent: MyOne Streptavidin C1 beads
labeling time: 15 min
replicate: Rep 1
|
| Growth protocol |
HeLa and HEK cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
FLAM-seq library preparation was prepared according to (Legnini et al., 2019). In brief, polyadenylated RNA was isolated from total RNA using Illumina Truseq mRNA preparation kit. A GI tail was appended to selected mRNA using tailing reagents from USB poly(A) length assay kit. Tailed RNA was purified using 1.8x RNAClean beads. Tailed RNA was reverse transcribed using SMARTScribe Reverse Transcriptase Kit and isoTSO and RT primer 1 or 2. cDNA was purified using 0.6x Ampure XP beads and PCR amplified using PCR primer 1 or 2 and PCR Primer IIA using Advantage 2 DNA polymerase mix. FLAM-seq libraries were purified 2x using 0.6x Ampure XP beads. PacBio adapters addition and sequencing was performed according to standard procedures.A detailed protocol for the FLAM-seq method can be found at https://protocolexchange.researchsquare.com/article/pex-398/v1
FLAM-seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel |
| |
|
| Data processing |
Circular Consensus Sequence (CCS) reads were generated within the SMRT Link browser 5.0 (minimum full pass of three and minimum predicted accuracy of 90).
Once the raw data have been processed, the final CCS fastq files can be used directly as input for the FLAM-seq analysis pipeline, available at: https://github.com/rajewsky-lab/FLAMAnalysis.
Genome_build: hg38 / mm10
Supplementary_files_format_and_content: Processed *_gene_polyA_length.csv files contain the extrac ted poly(A) tail length and sequence as well mapped gene locus for each read
|
| |
|
| Submission date |
Nov 10, 2021 |
| Last update date |
Jan 07, 2023 |
| Contact name |
Jonathan Alles |
| E-mail(s) |
Jonathan.Alles@mdc-berlin.de
|
| Organization name |
Max Delbruck Center Berlin
|
| Department |
Berlin Institute for Medical Systems Biology (BIMSB)
|
| Lab |
Nikolaus Rajewsky Lab
|
| Street address |
Hannoversche Straße 28
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL26180 |
| Series (1) |
| GSE188539 |
Rapid nuclear deadenylation of mammalian messenger RNA |
|
| Relations |
| BioSample |
SAMN23028438 |
| SRA |
SRX13096931 |
