|
| Status |
Public on Jul 19, 2024 |
| Title |
MNU_OVA_NG_FF_50pg_rep2 (rna-seq) |
| Sample type |
SRA |
| |
|
| Source name |
mouse normal urothelium (MNU)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: normal urothelium
rna seq library kit: Ovation SoLo NuGEN RNA-seq System (OVA-NG)
rna input quantity: 50pg
method of sample preservation: FF
|
| Treatment protocol |
The normal urothelium of each mouse was divided in two parts, one part was frozen in liquid nitrogen and the other part was conserved in a solution of buffered formalin for 24 hours. Formalin Fixed Paraffin Embedded (FFPE) samples were embedded into wax blocks. The fresh frozen (FF) specimens were conserved in a freezer until the RNA extraction. The mouse bladder cancer cell line (BC57) harvested cell pellet was divided in two parts and underwent the same protocol as the mouse normal urothelium.
|
| Growth protocol |
The mouse bladder cancer cells, BC57, have been culture in DMEM 10% fetal bobine serum. Nine mice were sacrificed and their normal urothelium were removed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from FFPE were extracted by using Qiagen RNeasy FFPE. Ten slices of five microns of thickness of FFPE samples were dewaxed using Deparaffinization solution and were extracted according to manufacturer instructions (RNeasy FFPE tissue Handbook). Total RNA from FF samples were extracted with Qiagen RNeasy mini according to manufacturer instructions.
Ovation SoLo NuGEN RNA-seq System: cDNA was synthesized and amplified from 50pg, 250pg, 2ng of total RNA from mouse normal urothelium and mouse bladder cancer cell line using the Ovation SoLo RNA-seq System (NuGEN) which allows to perform a strand specific RNA sequencing. Total RNA underwent a DNAse treatment then random priming allowed to first and the second strand cDNA synthesis end Illumina index was ligated. Before library amplification step, a qPCR was performed to determine the optimal number of library amplification cycles required for a given input and sample type. The depletion of ribosomal sequences was done by the InDA-C technology. PCR amplification was finally achieved to create the final cDNA library. Sequencing was carried out using 2*100 cycles (paired-end reads, 100 nucleotides) on an Illumina HiSeq2000 instrument (high output flow cells) to get around 8M paired reads per sample.
SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian: cDNA was synthesized and amplified from 250pg and 2ng of total RNA from mouse bladder cancer cell line and mouse normal urothelium. This kit uses the SMART technology for Switching Mechanism at 5' End of RNA Template to allow a strand specific RNA sequencing. Total RNA underwent a DNAse treatment and converted to cDNA by SMARTScribe Reverse Transcriptase. When SMARTScribe Reverse Transcriptase reached the 5’ end of RNA fragment, its terminal transferase activity added a few non-templated nucleotides to the 3’ end of the cDNA. The non-templated nucleotides were uses as a primer for the addition of Illumina index adapters through PCR. Next, the ribosomal depletion was carried out by ZapR enzyme, in presence of specific probes recognizing rRNA sequences. PCR amplification was finally achieved to create the final cDNA library. Sequencing was carried out using 2*100 cycles (paired-end reads, 100 nucleotides) on an Illumina HiSeq2000 instrument (high output flow cells) to get around 11M paired reads per sample.
Illumina TruSeq Stranded mRNA: total RNA from FF samples with a RIN > 7 were used for sequencing. RNA sequencing libraries were prepared from 1µg of total RNA from mouse bladder cancer cell line and mouse normal urothelium using Illumina TruSeq Stranded mRNA which allowed to perform a strand specific RNA sequencing. A first step of poly(A) selection using magnetic beads was done to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing and then ligated to the TruSeq indexed adapters. PCR amplification was finally achieved to create the final cDNA library. After qPCR quantification, sequencing was carried out using 2*100 cycles (paired-end reads, 100 nucleotides) on an Illumina HiSeq2000 instrument (high output flow cells) to get around 60M paired reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
expCount_raw_count_nugen.txt
expCount_norm_nugen.txt
A574T43
|
| Data processing |
Reads were aligned to mouse reference genome (mm10) by STAR using ENCODE parameters
Bam files were indexed by samtools
Read counting was done by the function featureCounts from Rsubreads.
Genome_build: mm10
Supplementary_files_format_and_content: .txt files represent the raw counts of each genes generated by featuresCount and the upper quartile normalized count.
|
| |
|
| Submission date |
Jul 25, 2018 |
| Last update date |
Jul 19, 2024 |
| Contact name |
Jennifer Wong |
| Phone |
689153887
|
| Organization name |
Institue Curie
|
| Department |
UMR144
|
| Lab |
Molecular oncology
|
| Street address |
12 rue lhomond
|
| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE117677 |
Influence of low-input RNA from FF and FFPE samples on gene expression quantification by NanoString, microarray and RNA-seq: a comparative study [RNA-seq] |
| GSE117678 |
Influence of low-input RNA from FF and FFPE samples on gene expression quantification by NanoString, microarray and RNA-seq: a comparative study |
|
| Relations |
| BioSample |
SAMN09716611 |
| SRA |
SRX4459294 |
