|
| Status |
Public on Jun 05, 2015 |
| Title |
KO_Ctrl_Pulldown_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
KO_Ctrl_Pulldown
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: human embryonic kindey cells
enrichment: N3-CMC treat and pull down
|
| Growth protocol |
cells was maintained in DMEM with 10% FBS and penicillin/streptomycin.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
total RNA was isolated from cell lines or tissues with Trizol(invitrogen). 10 ug mRNA was enriched from 1 mg total RNA with two rounds of polyA selection using Dynabeads Oligo(dT)25 (invitrogen) and one round of rRNA depletion using Ribominus Transcriptome kit (invitrogen).
The Biotin-CMC labeling of ψ was performed as previously described with some modification. The mRNA was was added to 0.2 M Chemically-synthesized N3-CMC in BEU buffer followed by incubated at 37 °C for 20 minutes. The Chemically-synthesized N3-CMC was removed by ethanol precipitation. The RNA pellet was dissolved in sodium carbonate buffer and incubated at 37°C for 6h to remove N3-CMC RNA was purified by ethanol precipitation and dissolved in H2O. Click reaction was performed with addition of 20 mM DBCO-(PEG)4-biotin (sigma) to a final concentration of 150 μM followed by incubating at 37°C for 2h.The mock sample was treated as above only without addition of Chemically-synthesized N3-CMC.The biotin-CMC-Ψ RNA was enriched by Streptavidin C1 Dynabeads (Invitrogen).The library construction was performed according to the iCLIP library construction protocol with some modifications. For dephosphorylation of 3'ends, RNA were treated with rSAP (NEB) and incubated at 37 °C for 30 minutes.RNA linker ligation was performed with T4 RNA ligase 1 (NEB) at 16 °C overnight.Reverse Transcription was performed with Superscript III reverse transcriptase (Invitrogen). The cDNA was purified with 6% UREA-PAGE to remove excessive RT primers. Purified cDNA was circligated with CircLigase II (Epicentre) followed by ethanol precipitation. Linearization of cDNA was performed with annealing with a cut oligo and followed by BamHI digestion. The cDNA was amplified by PCR with primers. PCR products were purified by 8% TBE gel and sequenced on Illumina Hiseq2000 or Hiseq2500 with single end reads (50bp).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
barcode: NNNNGTACNN
|
| Data processing |
library strategy: CAP-seq
Tirm_galore[version 0.3.7, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/] was used to remove or trim adaptor contained reads and low quality reads, requiring the minimum length of trimmed reads to be 20 bases. Artificial sequences, two bases added during library construction at the 5 prime of reads, were also removed
Remained reads were mapped to the human or mouse transcriptome (downloaded from UCSC genome tables) using BWA[0.7.5a-r405, Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25:1754-60. [PMID: 19451168]]
Genome_build: hg19, mm9
Supplementary_files_format_and_content: txt format; containing 7 columns are: 1)mRNA 2)Position 3)Base on this position 4)Reads with mapping position start at the (Pos+1) 5)Reads covered this position 6) sum of (column 4 and 5) 7)Stop rate estimated as column 4 devided by column 6
|
| |
|
| Submission date |
Nov 26, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ping Zhu |
| E-mail(s) |
[email protected]
|
| Organization name |
Institute of Hematology and Blood Disease Hospital
|
| Department |
State Key Laboratory of Experimental Hematology
|
| Street address |
288, Nanjing Road, Heping District
|
| City |
Tianjin |
| ZIP/Postal code |
300020 |
| Country |
China |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE63655 |
Chemical Pull-Down Reveals Comprehensive and Dynamic Pseudouridylation in Mammalian Transcriptome |
|
| Relations |
| BioSample |
SAMN03222674 |
| SRA |
SRX768638 |
