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Hi All,
I submitted 12 samples for ChIP-seq using the NexSeq Platform. Briefly, these samples represent 3 ChIP Biological Replicates:
Mock
Transcription Factor (TF)
Histone H3 (CT)
Input
According to the TapeStation, ChIPed DNA from Mock and TF were pretty low (500 pg total DNA), therefore, I decided to use Kapa Hyper Prep Kit. Barcodes (No.1 to 12) were from Bioo Scientific.
All samples were normalized to 500 pg initial DNA template, 19 PCR cycles.
After size selection (AMPure beads), samples were subjected to TapeStation. Average of DNA fragments ranged from 300-400 bp across all samples. We then were recommended the NextSeq platform, 75-bp, single end features. All samples were normalized to 6 nM and pooled in 20 microliters. We used the whole flow cell. After getting the raw data back, 11 samples contained poly AT-stretches, likely to be artifacts, only one Mock sample contained a low percentage of reads with those AT-stretches. Basically, 75% of the whole raw data contained such AT-stretches. I wonder if these artifacts were created during the library preparation or the NextSeq platform could introduce them. I'm attaching a PDF file showing the electropherogram of one sample and the AT-stretches present in our raw reads. Thanks a lot for the insight!
Miguel
I submitted 12 samples for ChIP-seq using the NexSeq Platform. Briefly, these samples represent 3 ChIP Biological Replicates:
Mock
Transcription Factor (TF)
Histone H3 (CT)
Input
According to the TapeStation, ChIPed DNA from Mock and TF were pretty low (500 pg total DNA), therefore, I decided to use Kapa Hyper Prep Kit. Barcodes (No.1 to 12) were from Bioo Scientific.
All samples were normalized to 500 pg initial DNA template, 19 PCR cycles.
After size selection (AMPure beads), samples were subjected to TapeStation. Average of DNA fragments ranged from 300-400 bp across all samples. We then were recommended the NextSeq platform, 75-bp, single end features. All samples were normalized to 6 nM and pooled in 20 microliters. We used the whole flow cell. After getting the raw data back, 11 samples contained poly AT-stretches, likely to be artifacts, only one Mock sample contained a low percentage of reads with those AT-stretches. Basically, 75% of the whole raw data contained such AT-stretches. I wonder if these artifacts were created during the library preparation or the NextSeq platform could introduce them. I'm attaching a PDF file showing the electropherogram of one sample and the AT-stretches present in our raw reads. Thanks a lot for the insight!
Miguel
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