I did not perform Fragmentation for my 1st FFPE sample. Now I am thinking I may need do to do that to narrow the insert size ...
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Originally posted by HedleyC View PostJakob, I have been involved in a piece of work trying to do the same thing - but we just got massive adapter contamination in the final data. What size range was your FFPE RNA peak after RiboZero GOLD but before the ScriptSeq? Ours was between 25-500bp approximately, peak generally around 100.
Regards,
Hedley
/Jakob
Comment
-
Originally posted by JakobHedegaard View PostRNA size range as yours - but with a sligthly higher peak ~200 nt. How do you purify the library after amplification? Using MiniElute will give you some "primer-dimer" contamination. We are using AMPure XP but you may need to play around with the bead ratio to get rid of the "primer-dimer" without removing too much of the library. Have you considered gel purification?
/Jakob
The ScriptSeq and later steps were performed by a 3rd party. They used AMPure XP, but it's unclear if they used the right bead ratio to minimise carry-through of the adapter dimers (I suspect not). We are doing some repeat work and also looking at another FFPE kit, so if I find out anything useful, will post it here at some point.
/Hedley
Comment
-
@ServiceXS, we obtained enough (>500 ng total RNA) purifiying from 2-3 sections from a FPFE block. For Ribo-Zero we started with 500 ng total RNA but have prepared libraries with 100 ng RNA - but with more variation in quality and yield.
@woodydon, using Ribo-Zero will result in a much more complex library than using poly-dA selected RNA. Depending on the sample conditions (tissue, stage, etc.) some transcripts can dominate the profile - and this applies to both poly-dA and Ribo-Zero treated RNA. Analysing data from Ribo-Zero treated RNA, I think the major challenge is the profiling of both mature and pre-spliced transcripts which introduce some kind of bias.
Cheers, /Jakob
Comment
-
@ServiceXS, we obtained enough (>500 ng total RNA) purifiying from 2-3 sections from a FPFE block. For Ribo-Zero we started with 500 ng total RNA but have prepared libraries with 100 ng RNA - but with more variation in quality and yield.
@woodydon, using Ribo-Zero will result in a much more complex library than using poly-dA selected RNA. Depending on the sample conditions (tissue, stage, etc.) some transcripts can dominate the profile - and this applies to both poly-dA and Ribo-Zero treated RNA. Analysing data from Ribo-Zero treated RNA, I think the major challenge is the profiling of both mature and pre-spliced transcripts which introduce some kind of bias.
Cheers, /Jakob
Comment
Latest Articles
Collapse
-
by seqadmin
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...-
Channel: Articles
Yesterday, 07:01 AM -
-
by seqadmin
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
58 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
54 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
45 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
55 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment