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Old 05-27-2013, 05:52 AM   #1
chiaraf
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Default cDNA as starting material for TruseqDNA

Hi,
I hope that someone can help me!
I'd like to do whole genome reseq from cDNA double strand using the truseqDNA kit from Illumina.
Do I need to change the protocol for Covaris fragmentations because of cDNA starting material instead of genomic DNA?
Many thanks!
Chiara
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Old 06-01-2013, 02:00 PM   #2
Amy0615
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Smile similar questions

I am doing some RNA-seq and I wonder whether it is possible to use cDNA after 1st stand synthesis as input using the illumina DNA prep kit?
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Old 06-03-2013, 11:37 AM   #3
barrmur
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You could use cDNA in the truseq prep but after fragmentation there might not be much left, depends on your input. Lots of people have used nextera sample preps. Lower sample input and no need to fragment using covaris. Transposon mediated fragmentation is active on cDNA.

Hope this helps.
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Old 06-04-2013, 03:09 AM   #4
chiaraf
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last week I fragmented the cDNA with Covaris and then I started to prepare libreries with the truseqDNA...libreries are fine but have a mean fragment size of 250 bp with adapter...
I'll have the sequencing reads overlapped with a 100PE and I know it's not good for bioinfo analysis..Next time I'll try with nextera as suggested by barrmur.
many thanks,
Chiara
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Old 06-05-2013, 05:58 AM   #5
microgirl123
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Hi Chiaraf, what did you use for your settings on the Covaris?
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Old 06-05-2013, 06:27 AM   #6
chiaraf
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Hi microgirl123,
I used the following protocol:
Peak IP 175
Duty factor 10%
Cycles / burst 200
Treament Time 120 sec
Temperature 7C
Water level 12
Sample Volume 50 ul
This is the protocol suggested in the Ovation RNA-Seq System V2 - NuGEN (That was the kit we used to obtain cDNA DS from RNA.
Chiara
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Old 06-05-2013, 06:41 AM   #7
microgirl123
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I'm not a Covaris expert and can't remember what everything means (maybe someone else can chime in) but I think your fragments are smaller than you'd like because you're oversonicating. I generally get ~500 bp fragments (so library size with adapters is ~650 bp) using a peak IP of 175, duty factor of 5%, 200 cycles/burst, and a time of 55 seconds.

We don't use the TruSeq DNA kit (we have an IntegenX Apollo robot and use their kits) - you may have to play around with their chemistry to select the larger size fragments.

ETA: this is for a 50 ul sample
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Old 06-05-2013, 06:49 AM   #8
chiaraf
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Dear microgirl123,
you're right ...probably I oversonicating..
Many thanks for your help!!
Chiara
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Old 06-11-2013, 02:54 AM   #9
addyblanch
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Just to add, settings are different depending on which covaris you use.
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Old 06-11-2013, 05:02 PM   #10
magnolia93
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Quote:
Originally Posted by Amy0615 View Post
I am doing some RNA-seq and I wonder whether it is possible to use cDNA after 1st stand synthesis as input using the illumina DNA prep kit?
Not after first strand synthesis, but after second strand synthesis. You cannot ligate to the RNA/DNA hybrid.
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Old 06-12-2013, 10:52 AM   #11
rthornton4
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We have been generating cDNA using the NuGEN Ovation kit and putting it into the Illumina DNA-Seq kit with no problems. We are beginning to adjust the settings on the Covaris to get larger cDNA fragments before library construction. It works very well.
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