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=== Discussion === * From the result, I could not distinguish the reactions between 1nu and 0nu as I saw a very strong smear along the lane. * I saw stronger signal in 10nu and 100nu. However the background was still very high and the pattern looked different from RRBS sequencing libraries. In addition, the range of sequencing libraries was too board to select for sequencing. * Youjin noticed strong signal at ~200bp or below in some samples, and he was interested to do sequencing on those samples that has been cropped in the gel images. However, for me, it's very difficult to interpret the result. I ==== What I have learnt from this experiment ==== * When first PCR was allowed to saturate, it generate very strong background a long the lane in all samples. This background might come from adaptors. * After 2 round of bead purification, it can remove only the signal below 150bp or so. However the smear along the lane is still there. This background will interfere 2nd PCR, so we would see it getting saturated at very early cycle number (6cycles). These background overcomes the real signal of library from the cell. * To resolve this, I plan to repeat experiment and tried to follow what I have done before like using TruSeq adaptor. This make me easy when set up PCR and follow incubation condition for ligation reaction. * The most important thing I would do is that I will monitor qPCR of 1st PCR and stopped once get close to the plateau. This will prevent the background from over amplified of adaptor dimers. This may be risky if the PCR cycle number is too small to amplify enough signal of the real template. I could lose a lot of PCR product of the real signal by performing 2 rounds of bead purification. * I would not say that this experiment totally failed, but the sub optimized condition of PCR cycle number in 1st PCR that made the background was too high.
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