Different cell-lines giving different levels of uniquely mapped reads?
Hi all,
We are carrying out some RNA-Seq on the full transcriptome of a cell line (x) and a cell line derived xenograft (y). RNA was harvested from x, half of it was taken and heat treated to degrade the RNA, bringing the RIN from 9.5 to 4.5. A freshly frozen CDX sample from y and an FFPE CDX sample for y also had their RNA harvested; the RIN was 9.5 and 2.5 respectively.
To quickly summarise:
Cell line (x): RIN 9.5 and 4.5
CDX (y): RIN 9.5 and 2.5
All 4 samples were put through three different library prep protocols (skipping any RNA degradation steps for the degraded samples) and then sequenced. Upon alignment analysis, both samples from x have more 'uniquely mapped reads' and less 'unmapped: too short' reads, with about the same number of 'mapped to too many loci' reads. Alignment of the degraded x RNA sample also seems to be better than the intact y RNA sample...
The quality of alignment seems to be much better with samples from x in all three protocols, despite being treated the same as the other samples. I'm fairly certain that the DV200 score was also similar throughout all samples before library prep. What could be causing this?
It might be worth adding that the cell line is a cancer cell line and the CDX is a cancer biopsy (derived from a different cell line). Could the differential gene expression have some effect?
We are carrying out some RNA-Seq on the full transcriptome of a cell line (x) and a cell line derived xenograft (y). RNA was harvested from x, half of it was taken and heat treated to degrade the RNA, bringing the RIN from 9.5 to 4.5. A freshly frozen CDX sample from y and an FFPE CDX sample for y also had their RNA harvested; the RIN was 9.5 and 2.5 respectively.
To quickly summarise:
Cell line (x): RIN 9.5 and 4.5
CDX (y): RIN 9.5 and 2.5
All 4 samples were put through three different library prep protocols (skipping any RNA degradation steps for the degraded samples) and then sequenced. Upon alignment analysis, both samples from x have more 'uniquely mapped reads' and less 'unmapped: too short' reads, with about the same number of 'mapped to too many loci' reads. Alignment of the degraded x RNA sample also seems to be better than the intact y RNA sample...
The quality of alignment seems to be much better with samples from x in all three protocols, despite being treated the same as the other samples. I'm fairly certain that the DV200 score was also similar throughout all samples before library prep. What could be causing this?
It might be worth adding that the cell line is a cancer cell line and the CDX is a cancer biopsy (derived from a different cell line). Could the differential gene expression have some effect?