GSM3307128: MNU_OVA_NG_FF_2ng_rep2 (rna-seq); Mus musculus; RNA-Seq - SRA

SRX4459290: GSM3307128: MNU_OVA_NG_FF_2ng_rep2 (rna-seq); Mus musculus; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 3.5M spots, 713.7M bases, 332.2Mb downloads

Submitted by: NCBI (GEO)

Study: 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]

PRJNA482872 • SRP155276 • All experiments • All runs

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Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For RNA-sequencing, each sample was tested by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) . Results obtained with the two tested kits were compared to those based on poly(A) tail enrichment. To determine which is the best kit suitable for RNA-seq, low-input and low-quality RNA samples, we performed RNA-seq control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, library kit and method of sample preservation (FF or FFPE). Results: The Ovation SoLo NuGEN RNA-seq System library kit are recommended for quantification of gene expression of FFPE and FF samples at low-input (down to 50pg) whereas the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian is only suitable for FF samples from 250pg of total RNA. Overall design: For RNA-sequencing, the 4 samples were tested in triplicate by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) .

Sample: MNU_OVA_NG_FF_2ng_rep2 (rna-seq)

SAMN09716615 • SRS3596611 • All experiments • All runs

Organism: Mus musculus

Library:

Instrument: Illumina HiSeq 2000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: PAIRED

Construction 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.

Experiment attributes:

GEO Accession: GSM3307128

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 3.5M spots, 713.7M bases, 332.2Mb

Run	# of Spots	# of Bases	Size	Published
SRR7594294	3,533,398	713.7M	332.2Mb	2024-07-19

ID:: 6044975

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