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Scriptseq V2 : fragmentation problem ?
Hi,
We want to study the transcriptome of breast cancer cell lines and tissue, so we want to do RNA-seq experiment.
We prepare our sample with Tripure extraction, clean up (Qiagen), DNAse treatment. We check the quality with the Bioanalyser, our samples have a RIN of about 8 - 8,5.
Then we use the Ribozero kit with 2µg of total RNA as input. We use the clean and concentrator (Zymo) to clean our sample. We obtain about 12ng of ribodepleted RNA (0,6ng/µl in 20µl). We check our samples with the Bioanalyser (see http://www.fichier-pdf.fr/2012/01/11...bozero-090112/), everything seems ok.
Then we prepare the library (scriptseq V2) with 9,5 µl of our ribodepleted RNA, we exactly follow the protocol (We purify the cDNA with the MinElute PCR purification kit in step 4.D., and we purify the RNA-seq library using AMPure XP purification (step 4.F.) , and we perform 15 cycles for the PCR)
After that, we quantify with the Qubit, and we obtain +/- 3 to 5 ng/µl of DNA (total amount 60 to 100 ng, it's less than expected).
When we put 1µl of our sample on the Bioanalyser (DNA 1000), we see nothing, no peak, no concentration.
We tried one qPCR with this DNA (+/- 5ng DNA per well) , and we obtain good Cq values (we tried Actin (Cq = 17,2), 18S (Cq = 25,5), 28S (Cq = 22,1).
So we suppose that the fragmentation step didn't work well.
We did all the experiment twice, and we obtain similar result.
What do you think about that ?
Has someone else use this kit ? If yes, do you have any problem ?
Thank you
Best regards,
We want to study the transcriptome of breast cancer cell lines and tissue, so we want to do RNA-seq experiment.
We prepare our sample with Tripure extraction, clean up (Qiagen), DNAse treatment. We check the quality with the Bioanalyser, our samples have a RIN of about 8 - 8,5.
Then we use the Ribozero kit with 2µg of total RNA as input. We use the clean and concentrator (Zymo) to clean our sample. We obtain about 12ng of ribodepleted RNA (0,6ng/µl in 20µl). We check our samples with the Bioanalyser (see http://www.fichier-pdf.fr/2012/01/11...bozero-090112/), everything seems ok.
Then we prepare the library (scriptseq V2) with 9,5 µl of our ribodepleted RNA, we exactly follow the protocol (We purify the cDNA with the MinElute PCR purification kit in step 4.D., and we purify the RNA-seq library using AMPure XP purification (step 4.F.) , and we perform 15 cycles for the PCR)
After that, we quantify with the Qubit, and we obtain +/- 3 to 5 ng/µl of DNA (total amount 60 to 100 ng, it's less than expected).
When we put 1µl of our sample on the Bioanalyser (DNA 1000), we see nothing, no peak, no concentration.
We tried one qPCR with this DNA (+/- 5ng DNA per well) , and we obtain good Cq values (we tried Actin (Cq = 17,2), 18S (Cq = 25,5), 28S (Cq = 22,1).
So we suppose that the fragmentation step didn't work well.
We did all the experiment twice, and we obtain similar result.
What do you think about that ?
Has someone else use this kit ? If yes, do you have any problem ?
Thank you
Best regards,
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