|
| Status |
Public on Mar 14, 2017 |
| Title |
PBT003 gliblastoma stem cells, control shRNA, Input |
| Sample type |
SRA |
| |
|
| Source name |
PBT003 gliblastoma stem cells treated with virus expressing control shRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Brain tumor PBT003 gliblastoma stem cells
molecule purification: poly(dT) selected RNA, fragmented
|
| Treatment protocol |
Cells were treated with lentivurs expressing control shRNA or target (METTL3 or METTL14) shRNA for 24 hours and were cultured for another 7 days before RNA extraction.
|
| Growth protocol |
Cells were cultured in DMEM-F12 medium supplemented with B27 and growth factors EGF and FGF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by Trizol (Ambion) according to the manufacturer's instructions. mRNA was then purified using Dynabeads mRNA purification kit (Ambion, Catalog # 61006).
RNA fragmentation was performed by sonication at 10 ng μl-1 in 100 μl RNase-free water using Bioruptor Pico (Diagenode) with 30 s on / 30 s off cycle for 30 cycles. 5% of the fragments was saved as input. m6A-immunoprecipitation (m6A IP) and library preparation were performed according to a published protocol (Dominissini et al., 2013). In detail, 2.5 μg affinity purified anti-m6A rabbit polyclonal antibody (Synaptic Systems; Catalog # 202003) and 20 μl Protein A beads (ThermoFisher; Catalog# 10002D) were used for each affinity pull-down. m6A antibody-bound RNAs were eluted with 100 μl elution buffer and recovered by RNA Clean and Concentrator-5 (Zymo), and subjected to RNA library preparation with TruSeq Stranded mRNA Library Prep Kit.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
RNA-seq data (input RNA) of PBT003 gliblastoma stem cells treated with virus expressing control shRNA
ShControl.R1.bed
|
| Data processing |
All samples were sequenced by Illumina Hiseq 4000 with single-end 50-bp read length. The deep sequencing data were mapped to human genome version 38 (GRCh38).
Data analysis for each experiment: (1) for m6A-seq, reads were aligned to the reference genome using Tophat v2.0.14 with parameter -g 1 --library-type=fr-firststrand. RefSeq Gene structure annotations were downloaded from UCSC Table Browser. The longest isoform was used if the gene had multiple isoforms. Aligned reads were extended to 150 bp (average fragments size) and converted from genome-based coordinates to isoform-based coordinates, in order to eliminate the interference from introns in peak calling. The peak calling method was modified from published work (Dominissini et al., 2012). To call m6A peaks, the longest isoform of each gene was scanned using a 100 bp sliding window with 10 bp step. To reduce bias from potential inaccurate gene structure annotation and the arbitrary usage of the longest isoform, windows with read counts less than 1/20 of the top window in both m6A-IP and input sample were excluded. For each gene, the read counts in each window were normalized by the median count of all windows of that gene. A Fisher exact test was used to identify the differential windows between IP and input samples. The window was called as positive if the FDR < 0.01 and log2(Enrichment Score) >= 1. Overlapping positive windows were merged. The following four numbers were calculated to obtain the enrichment score of each peak (or window): reads count of the IP samples in the current peak/window (a), median read counts of the IP sample in all 100 bp windows on the current mRNA (b), reads count of the input sample in the current peak/window (c), and median read counts of the input sample in all 100 bp windows on the current mRNA (d). The enrichment score of each window was calculated as (a×d)/(b×c). (2) for mRNA-seq (the input samples of m6A-seq), reads were mapped with Tophat and Cufflink (v2.2.1) was used to calculate the FPKM of each gene to represent their mRNA expression level.
Genome_build: hg38
Supplementary_files_format_and_content: m6A peak list from IP samples
|
| |
|
| Submission date |
Feb 13, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
zhike lu |
| E-mail(s) |
zhikelu@gmail.com
|
| Organization name |
University of Chicago
|
| Department |
department of chemistry
|
| Street address |
5801 South Ellis Avenue
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE94808 |
m6A-seq in glioblastoma stem cells |
|
| Relations |
| BioSample |
SAMN06324080 |
| SRA |
SRX2554929 |
