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Forum: Sample Prep / Library Generation 03-13-2012, 09:14 AM
Replies: 1
Views: 1,756
Posted By DrDTonge
Question Pippin Prep - cDNA musings...

Dear all,

We've purchased a Pippin Prep and are awaiting training to use the 3% for size selecting our small RNA libraries. Briefly, these comprise of 18 - 38nt inserts with adapters making a...
Forum: Bioinformatics 01-12-2012, 11:20 PM
Replies: 0
Views: 2,668
Posted By DrDTonge
Forum: SOLiD 08-25-2011, 12:14 AM
Replies: 0
Views: 1,870
Posted By DrDTonge
SOLiD Adapter Sequences

Dear all,

Having run and analyzed a 12 sample multiplexed RNA-Seq study, I have noted that some of the samples are under represented (total reads are very low).

Moreover, I've noted that the...
Forum: Bioinformatics 07-26-2011, 11:43 PM
Replies: 4
Views: 3,014
Posted By DrDTonge
I'm doing it in colour-space using CLC Bio...

I'm doing it in colour-space using CLC Bio...
Forum: Bioinformatics 07-26-2011, 12:31 AM
Replies: 4
Views: 3,014
Posted By DrDTonge
or the reverse complement CTGCTGTACGGCCAAGGCG

or the reverse complement

CTGCTGTACGGCCAAGGCG
Forum: Bioinformatics 07-26-2011, 12:11 AM
Replies: 4
Views: 3,014
Posted By DrDTonge
SOLiD Barcoded Adapter Trimming

Dear all,

I've run a barcoded SOLiD run with a set of 12 small RNA samples. Following the sequencing run, I've performed the primary analysis and downloaded the csfasta (F35) and qual files.

On...
Forum: SOLiD 07-11-2011, 07:52 AM
Replies: 2
Views: 1,409
Posted By DrDTonge
Much appreciated. D

Much appreciated.

D
Forum: SOLiD 07-11-2011, 07:16 AM
Replies: 2
Views: 1,409
Posted By DrDTonge
Confused...

Dear all,

I've run the SOLiD small RNA workflow (35bp reads) and have just downloaded my reads from the server following primary analysis (it was a barcoded run).

My confusion is that my...
Forum: Bioinformatics 07-11-2011, 04:39 AM
Replies: 1
Views: 1,543
Posted By DrDTonge
Small RNA - Reads too long after trimming

I have run a batch of small RNA samples on the SOLiD system. Following sequencing, I've trimmed the adapters (in colour space) using CLC Bio.

The statistics are as follows...

Reads Trimmed:...
Forum: Bioinformatics 07-04-2011, 02:30 AM
Replies: 0
Views: 1,342
Posted By DrDTonge
RNA2MAP (SOLiD)

Dear all,

I've currently got a set of small RNA samples on the SOLiD 4 and am wanting to perform some small RNA analysis.

My samples are canine and therefore I have obtained the cfa.gff file...
Forum: Sample Prep / Library Generation 06-29-2011, 11:10 PM
Replies: 6
Views: 2,345
Posted By DrDTonge
Yeah, I'll second that. Most certainly adapter...

Yeah, I'll second that. Most certainly adapter dimers from Agilent trace. Very interesting point raised by whw...I'll look into this for any future preps.

D
Forum: Sample Prep / Library Generation 06-20-2011, 02:55 AM
Replies: 0
Views: 1,285
Posted By DrDTonge
SOLiD Small RNA - Minimum % miRNA in library

Dear all,

I've prepared several small RNA libraries and have just this morning QC'd them to assess the % miRNA (120-130bp as a % of 25-150bp).

My question is how stringent is the recommendation...
Forum: Sample Prep / Library Generation 06-13-2011, 12:06 AM
Replies: 6
Views: 2,345
Posted By DrDTonge
Sorry the above was a rather vague (pre-coffee...

Sorry the above was a rather vague (pre-coffee infusion) answer....!

By no success I mean on running the re-amplified product against the original product (Agilent DNA 1000) there was no increase...
Forum: Sample Prep / Library Generation 06-12-2011, 11:54 PM
Replies: 6
Views: 2,345
Posted By DrDTonge
Dear all, Just for future reference, I've...

Dear all,

Just for future reference, I've tried the above technique (re-amplifying a small amount of the amplified cDNA using SOLiD 5' and 3' BC in an attempt to increase the % of miRNAs with...
Forum: Sample Prep / Library Generation 06-10-2011, 04:36 AM
Replies: 6
Views: 2,345
Posted By DrDTonge
Small RNA Library Question - SOLiD

Dear all,

So I've prepared my first smallRNA library from 4 fibrotic skins samples and 1 plasma sample. Following reverse transcription and amplificiation, I have run an Agilent DNA chip to...
Forum: Sample Prep / Library Generation 06-06-2011, 06:13 AM
Replies: 3
Views: 1,849
Posted By DrDTonge
Thanks for your response. Given the large...

Thanks for your response.

Given the large variation between samples (this is expected as they are from different disease stages) I am going to start with a standard volume of RNA (3ul) and then...
Forum: Sample Prep / Library Generation 06-06-2011, 02:44 AM
Replies: 3
Views: 1,849
Posted By DrDTonge
Sorry guys...any thoughts?

Sorry guys...any thoughts?
Forum: Sample Prep / Library Generation 06-02-2011, 11:54 PM
Replies: 3
Views: 1,849
Posted By DrDTonge
Normalisation

Dear all,

Apologies if this is a naive question however I'm just wondering at what stage people implement normalisation steps into their library preparation.

As a bit of background, I have 30...
Forum: Sample Prep / Library Generation 06-02-2011, 06:46 AM
Replies: 3
Views: 2,682
Posted By DrDTonge
Thanks very much for your input. I have run...

Thanks very much for your input.

I have run a trial DNase treatment and am unhappy with the recovery of small RNAs following precipitation.

Your comments with regards incomplete DNase digestion...
Forum: Sample Prep / Library Generation 06-02-2011, 12:43 AM
Replies: 3
Views: 2,682
Posted By DrDTonge
Going to give the DNase treatment a shot with a...

Going to give the DNase treatment a shot with a single sample to determine the RNA recovery post precipitation. I think I'd be happier running DNase prior to library prep so here goes...
Forum: Sample Prep / Library Generation 06-01-2011, 07:06 AM
Replies: 3
Views: 2,682
Posted By DrDTonge
DNase treat RNA before SOLiD Small RNA?

Dear all,

As the title suggests I am running a batch of small RNA sequencing using the SOLiD system. Having extracted good quality total RNA (RIN >9), the yield of some samples is rather low (as...
Forum: Sample Prep / Library Generation 05-09-2011, 11:56 PM
Replies: 8
Views: 5,196
Posted By DrDTonge
Dear Aushev, Many thanks for your response....

Dear Aushev,

Many thanks for your response. I agree, the higher concentrations are likely a result of inaccurate quantification although it's always nice to hear someone else is getting similar...
Forum: Sample Prep / Library Generation 05-05-2011, 01:20 AM
Replies: 8
Views: 5,196
Posted By DrDTonge
miRNA Extraction from Plasma

Dear all,

I'm currently working on the isolation of miRNA from canine and human plasma samples. Using the Qiagen column based protocol, I have been able to isolate around 300pg/ul of miRNA (based...
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