|
| Status |
Public on Apr 19, 2010 |
| Title |
10ng_library_methB_S2_2.5%ERCC_phaseV_pool15_mRNA |
| Sample type |
SRA |
| |
|
| Source name |
Drosophila S2 cell and ERCC RNA controls, 10ng
|
| Organisms |
Drosophila melanogaster; synthetic construct |
| Characteristics |
cell line: S2
external rna control: ERCC phase V pool 15
rna quantity: 10ng
library preparation method: B
|
| Growth protocol |
Drosophila S2 cells were grown at 25°C in Schneider's Drosophila Medium (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine serum (SAFC Biosciences, Lenexa, KS) and Penicillin-Streptomycin. S2 cell mRNA aliquots are from GSM410196.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from S2 cells was extracted using Trizol (Invitrogen, Carlsbad, CA), followed by mRNA isolation using the Oligotex poly(A) extraction kit (Qiagen, Valencia CA).
A master mRNA mixture was made from 300ng S2 mRNA and 7.5ng ERCC phase V pool15 RNA controls and serially diluted. Libraries were prepared according to GSM410196 with two variations. In method-A, size selection preceeded PCR amplification. In method-B, PCR amplification preceeded size selection. The library concentration of sample 100ng_library_methA_S2_2.5%ERCC_phaseV_pool15mRNA was measured by Nanodrop spectrophotometer and the concentration of all other libraries were measured by RT-PCR using the concentration of sample 100ng_library_methA_S2_2.5%ERCC_phaseV_pool15mRNA as a reference. The primers used in the real-time PCR are identical to PCR amplification primers in the library construction. 10nm DNA from each library was used for clustering on the flow cell.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
S2 mRNA is the same sample as GSM410196. Sequences and abundance information of ERCC phase V pool 15 used in this study were provided separately with the supplementary files:
GSE20555_ERCC_phaseV_pool15_sequence.txt
GSE20555_ERCC_phaseV_pool15_abundance.txt
PCR amplification was done before size selection during library construction.
Run32-091022_s_4
|
| Data processing |
We retained reads that passed default quality filtering parameters in the Illumina pipeline (v1.4.0). We aligned reads passing quality filtering using Bowtie (v0.10.0; parameter -m 1 -v 2). Sequences of the ERCC RNAs (*ERCC_map.txt) were used as the alignment reference. Additionally, Drosophila genome sequence (BDGP release 5, dm3) and ERCC RNA sequences were combined into a reference (*ERCC_dm3_map.txt). We used unique mapped reads with less than 2 mismatches for analysis.
|
| |
|
| Submission date |
Feb 27, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Oliver |
| E-mail(s) |
briano@nih.gov
|
| Phone |
301-204-9463
|
| Organization name |
NIDDK, NIH
|
| Department |
LBG
|
| Lab |
Developmental Genomics
|
| Street address |
50 South Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL10119 |
| Series (1) |
| GSE20555 |
RNA-Seq on libraries made from serial dilutions of Drosophila melanogaster S2 cell mRNA and ERCC external RNA controls |
|
| Relations |
| Reanalyzed by |
GSM3274604 |
| SRA |
SRX018873 |
| BioSample |
SAMN00010995 |
