After finishing the final set of eight MBD selections on 20201103, I’m finally ready to make the BSseq libraries using the Pico Methyl-Seq Library Prep Kit (ZymoResearch) (PDF). I followed the manufacturer’s protocols with the following notes/changes (organized by each section in the protocol):
GENERAL
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Protocol was followed for using input DNA range 1ng - 50ng.
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All thermalcycling was performed on the Roberts Lab PTC-200 (MJ Research).
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All thermalcycling used a heated lid temp of 104oC, unless a different temp was specified in the protocol.
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All elution steps were performed with heated elution buffer (55oC).
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All index primers not included with the kit were a mix of the Illumina TruSeq P5 primer (SRID: 1733) and an Illumina TruSeq P7 index primer (see table at bottom of page). The mix consisted of 10uM each of P5 and P7 primers. See the Roberts Lab Primer Database (Google Sheet) for info on the primers.
SECTION 2
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Used 0.5mL PCR tubes, since 0.2uL tubes were not specified and the 0.5mL tubes are easier to handle/work with.
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PrepAmp Mix was prepared as a master mix and then distributed to samples as required
| PrepAmp_component | single_rxn_vol(uL) | num_rxns | total_vol(uL) |
|---|---|---|---|
| PrepAmp Buffer (5x) | 1 | 26 | 26 |
| PrepAmp Pre-mix | 3.75 | 26 | 97.5 |
| PrepAmp Polymerase | 0.3 | 26 | 7.8 |
SECTION 3
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Elutions consistently returned 1.5uL less volume than input (e.g 12uL input returned 10.5uL).
- This was also noted by Shelly when she utilized this kit previously.
SECTION 4
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Recovery from SECTION 3 elution was only 10.5uL (expected 11.5uL based on protocol), so added 1.5uL H2O to each sample.
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Based on input DNA range (1ng - 50ng), number of cycles was set to 8.
SECTION 5
- Anticipating the loss in elution volume, samples were eluted with 13.5uL in the preceding cleanup step and yielded 12uL (the target input volume for this section).
NOTE: Sample CH10-19 had a weird elution in SECTION 4 - only recovered 6.5uL. Brought volume up to 12uL with H2O for required input volume in SECTION 5.
Next step, run the samples on the Bioanalyzer for QC to see how they look.
Sample - Sequencing Primer Index Table
| Sample | Illumina_TruSeq_index_num | Illumina_TruSeq_Index_seq | SRID/ZymoID |
|---|---|---|---|
| CH01-06 | 1 | CGTGAT | 1732 |
| CH01-14 | 2 | CGATGT | A |
| CH01-22 | 3 | GCCTAA | 1731 |
| CH01-38 | 4 | TGACCA | B |
| CH03-04 | 5 | ACAGTG | C |
| CH03-15 | 6 | GCCAAT | D |
| CH03-33 | 7 | CAGATC | E |
| CH05-01 | 8 | TCAAGT | 1730 |
| CH05-06 | 9 | CTGATC | 1729 |
| CH05-21 | 10 | AAGCTA | 1728 |
| CH05-24 | 11 | GTAGCC | 1727 |
| CH05-26 | 12 | CTTGTA | F |
| CH07-06 | 13 | TTGACT | 1726 |
| CH07-11 | 14 | GGAACT | 1725 |
| CH07-24 | 15 | TGACAT | 1724 |
| CH09-02 | 16 | GGACGG | 1723 |
| CH09-11 | 17 | CTCTAC | 1722 |
| CH09-13 | 18 | GCGGAC | 1721 |
| CH09-28 | 19 | TTTCAC | 1720 |
| CH09-29 | 20 | GGCCAC | 1719 |
| CH10-01 | 21 | CGAAAC | 1718 |
| CH10-08 | 22 | CGTACG | 1717 |
| CH10-11 | 23 | CCACTC | 1805 |
| CH10-19 | 25 | ATCAGT | 1804 |
All sample processing info/history can currently be found here (Google Sheet):
Any additional project info will end up in this GitHub repo: