|
| Status |
Public on Jun 24, 2016 |
| Title |
GM12878 rep2 m6A pos |
| Sample type |
SRA |
| |
|
| Source name |
GM12878
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: GM12878
treatment: eluate (m6A positive) fraction of m6A-LAIC-seq
|
| Growth protocol |
cell culture: cultured on complete RPMI media supplemented with 15% FBS and 10% Pen/Strep antibiotic
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from cells according to manufacturer’s instructions using TRIzol LS reagent (Ambion). Total RNA was treated using DNAse I (Promega) for 20 minutes at 37ºC. The treated RNA was then acid phenol/chloroform extracted and chloroform extracted. The RNA was precipitated using 300mM final concentration of NaCl2 spiked with 1µl of 50mg/ml of Ultra Pure Glycogen (Promega) and 2.5 volume of 100% ethanol at -20ºC either for 2 hours or overnight. The precipitated RNA was then centrifuged using a refrigerated table-top at maximum speed (>13,000g) at 4ºC for 20 minutes. The precipitated RNA was then washed with 70ºC ethanol and centrifuged at maximum speed for an additional 10 minutes. The final pellet was then re-suspended in ultra pure H2O. PolyA RNA selection was performed twice using Dynabeads mRNA Purification Kit (Invitrogen Cat. #610.06) according to the manufacturer's protocol. The second polyA RNA selection was performed using the eluate of the first polyaA RNA selection as starting material according to the manufacture’s instruction.
100ng RNA were used for library construction and m6A-LAIC-seq using TrueSeq Stranded mRNA Sample Preparation Guide, entering the protocol by adding the Fragment, Prime, Finish Mix, skipping the elution step and proceeding immediately to the synthesis of the First Strand cDNA. From that point on, the exact steps of the Illumina TruSeq Stranded mRNA sample Preparation Guide were followed to the end.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
eluate (m6A positive) fraction of m6A-LAIC-seq
|
| Data processing |
We mapped strand-specific paired-end RNA-seq reads first to Ensembl genes and then to hg19 human genome incorporated with all spike-in sequences using tophat (v 1.4.1).
Only the properly paired and uniquely mapped reads were used in future analyses.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files were generated using BEDTools, the socres represent coverage of raw reads.
|
| |
|
| Submission date |
Feb 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jinkai Wang |
| Organization name |
University of California, Los Angeles
|
| Street address |
650 Charles E. Young Drive South, CHS 33-228
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-7278 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE66086 |
m6A level and isoform characterization sequencing (m6A-LAIC-seq) reveal the census and complexity of the m6A epitranscriptome |
|
| Relations |
| BioSample |
SAMN03354222 |
| SRA |
SRX883636 |
