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Forum: RNA Sequencing

09-22-2017, 08:04 AM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Yes, I plan to prepare all samples using the same...

Yes, I plan to prepare all samples using the same library prep methods.

Thanks very much for your feedback and helpful suggestions.

Forum: RNA Sequencing

09-21-2017, 07:16 AM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Thanks, jteeee2. I will definitely try this...

Thanks, jteeee2. I will definitely try this method with a lower SPRI clean-up ratio, as suggested by nucacidhunter. KAPA has always been a reliable NGS resource.

Forum: RNA Sequencing

09-21-2017, 05:41 AM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Should some form of fragmentation be performed...

Should some form of fragmentation be performed then? Does no fragmentation introduce bias by not capturing the larger mRNAs in the sample?

Forum: RNA Sequencing

09-21-2017, 04:28 AM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Given that the TruSeq kit is only capable of...

Given that the TruSeq kit is only capable of producing up to 200 bp inserts, and I intend to do a 2 x150 bp run, overlap is obviously unavoidable. I do not want to introduce bias in the data analysis...

Forum: RNA Sequencing

09-20-2017, 06:21 PM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Yes, annealing/extension is set to 45 sec. I...

Yes, annealing/extension is set to 45 sec.

I was under the assumption that final library insert size was dependent on fragmentation time and post-ligation size selection. Perhaps that is my...

Forum: RNA Sequencing

09-20-2017, 09:07 AM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Thanks for the input. I'm using the KAPA library...

Thanks for the input. I'm using the KAPA library quantification kit for qPCR.

I will try altering fragmentation time to 2 min at 94°C and doing a double-sided SPRI size selection for 350-500 bp...

Forum: RNA Sequencing

09-19-2017, 04:19 PM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

Thanks, nucacidhunter, I'm also concerned with...

Thanks, nucacidhunter, I'm also concerned with reading into the adapter or through the adapter into the flow cell and crashing the run; however, we have generated libraries using the "0 minute"...

Forum: RNA Sequencing

09-19-2017, 01:26 PM

Replies: 15

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

Views: 6,115

Posted By emily202

RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

I'm using the TruSeq Stranded Total RNA library preparation kit to generate libraries for 2x150 bp paired-end sequencing. Illumina's fragmentation protocol recommends 94°C for 8 minutes for intact,...

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