Inconsistent size distribution of fragmented mRNA and cDNA for RNA 3Seq

[boardlover]

Hello everyone,

I am new to the RNA 3' Sequencing experiment using a protocol developed in house based on Illumina TruSeq mRNA sample prep. The library, however, did not get amplified after many tries. So I started to impose quality control steps and see what went wrong.

The starting material is 6ug human total RNA, extracted 100-250ng mRNA. The mRNAs were then fragmented in 85C for 10 mins in the 5x First Stranded buffer from Superscript III kit. I took 2uL fragmented mRNA to run a Bioanalyzer RNA pico assay, which showed a peak around the lower range between 200-500 bp. The samples were then undergone cDNA synthesis (clean up by Qiagen kit), 3'A tailing (clean up by Qiagen kit), linker ligation (clean up by Ampure beads) and PCR (clean up by Ampure beads).

While mRNA fragmentation seemed to have acceptable pattern, all of a sudden there was a peak around 500-700 bp in cDNA and 3'A tailing product, but the the peak disappear in the linker ligation product. And even more strangely, most products from PCR was around 200-300 bp, back to expected. If you refer to the attached figure you know what I mean.

I discussed with my colleague who has experience in RNA Seq, and she said this was totally weird. Has anyone here ever looked at the mRNA and cDNA profiles in these intermediate steps? What are they supposed to look like? And what could have caused this strange phenomenon here? Desperate here! Any thoughts will be much appreciated!!!

[pmiguel]

I don't know what your peak is. But bioanalyzer DNA chips do weird things with single-stranded DNA/RNA. See here for more details. But the sizes you see will not be accurate if they are single stranded or contain lengths of single stranded areas.
--
Phillip

[boardlover]

But cDNA and the following should be double-stranded DNA?

I don't know what your peak is. But bioanalyzer DNA chips do weird things with single-stranded DNA/RNA. See here for more details. But the sizes you see will not be accurate if they are single stranded or contain lengths of single stranded areas.
--
Phillip

[cmbetts]

But cDNA and the following should be double-stranded DNA?

You only mentioned cDNA synthesis. Is this only first strand cDNA or is there a second-strand/polishing step that you didn't mention, because that would be your problem right there if it's only first strand...

Maybe if you posted a little more detail about your protocol it would be easier to diagnose?
Since you said it's 3' sequencing, I'm assuming the slightly more detailed protocol goes something like.

1) Purify mRNA by dT beads
2) Fragment RNA
3) First strand synthesis with a dT primer with Illumina read2 (or just P7)
4) Second strand synthesis
5) End Polishing/ A-tailing
6) Ligate Illumina Read1
7) PCR for indexing/completing any partial adapter sequences

Is that correct?

[pmiguel]

But cDNA and the following should be double-stranded DNA?

There would probably still be ssRNA around, unless RNAseH were used after first strand synthesis. Seems like that is pretty rare these days.

--
Phillip

[boardlover]

Thanks! The cDNA was after second strand synthesis.

Much appreciate your suggestion on posting the protocol in details:

1) Purified mRNA by dT beads (Dynabeads® mRNA Purification Kit)
2) Fragment RNA (85 degree 10 mins with R2SP Oligo dT, in 5X First-strand synthesis buffer from LifeTechnologies)
3) First-strand cDNA synthesis (DTT, dNTP, RNase Out, SuperScript III; 50 degree, 1 hour, 85 degree inactivation for 15 min)
4) Second strand synthesis (SS buffer, dNTP, E.coli DNA Ligase, E.coli DNA Polymerase I, E.coii RNase H; 16 degree 2 hours, then T4 DNA Polymerase for 30 mins; clean up with Qiagen MinElute Reaction Cleanup kit)
5) 3'A tailing (NEB buffer 2, dATP, Klenow Fragment, 3' to 5' exo; 37 degree 30 min; clean up with Qiagen MinElute Reaction Cleanup kit)
6) Linker ligation (Adaptor oligo mix from Illumina, DNA ligase buffer, DNA ligase; RT 20min; clean up with Ampure beads, 1:1)
7) PCR (P5-Rd1SP, P7-index-Rd2SP, Phusion PCR master mix; 98C 30s, 15 cycle of (98C/10s, 65C/30s, 72C/30s), 72C 5min; clean up with Ampure beads, 1:1)
Then run bioanalyzer.

I managed to make the first 3 libraries with pilot experiment with other people's reagents, but ever since got similar failed results many times after purchasing my own reagents. The item catalog numbers are the same, checked many times. Replaced enzymes and buffers already. Have no idea why this happened

You only mentioned cDNA synthesis. Is this only first strand cDNA or is there a second-strand/polishing step that you didn't mention, because that would be your problem right there if it's only first strand...

Maybe if you posted a little more detail about your protocol it would be easier to diagnose?
Since you said it's 3' sequencing, I'm assuming the slightly more detailed protocol goes something like.

1) Purify mRNA by dT beads
2) Fragment RNA
3) First strand synthesis with a dT primer with Illumina read2 (or just P7)
4) Second strand synthesis
5) End Polishing/ A-tailing
6) Ligate Illumina Read1
7) PCR for indexing/completing any partial adapter sequences

Is that correct?