Sam's Notebook

BS-seq Library Prep - C.gigas Larvae OA 1000ppm

by ["kubu4"] 1 min read

Bisulfite Conversion

Pooled 200ng each of the sheared 1B1 (4μL) & 1B2 (used the entire sample, 20μL) 5.13.11 1000ppm C.gigas larvae DNA samples for a total of 400ng. Total volume = 24μL.

Quantified the pooled DNA using the NanoDrop1000 (ThermoFisher) prior to initiating bisulfite conversion.

(http://eagle.fish.washington.edu/Arabidopsis/20150227_Emma_1000ppm_pool_preBS_plot.JPG)

Clearly, the NanoDrop measurements differ from the expected concentration. NanoDrop suggests the total amount of input DNA is ~1400ng (58ng/μL x 24μL = 1392ng). This is most likely due to RNA carryover, as DNA quantification using a fluorescence-based, double-stranded DNA assay performed previously shows a drastically lower concentration.

Proceeded with bisulfite conversion using the Methylamp DNA Modification Kit (Epigentek) in 1.5mL tube, according to the manufacturer’s protocol:

  • Added 1μL to sample, incubated 10mins @ 37C in water bath

  • Made fresh R1/R2/R3 solution (1.1mL R3 buffer added to vial of R2, vortexed 2mins, 40μL R1 added to mixture - Remainder stored @ -20C in “-20C Kit Components Box”)

  • Added 125μL of R1/R2/R3 solution to sample, incubated 90mins @ 65C in heating block with water

  • Addd 300μL R4 to sample, mixed, transferred to column, spun 12,000RPM 30s

  • Added 200μL R5 to column, spun 12,000RPM 30s

  • Added 50μL R1/ethanol solution to column, incubated 8mins @ RT, spun 12,000RPM 30s

  • Washed column with 200μL of 90% EtOH, spun 12,000RPM 30s; repeated one time.

  • Eluted DNA with 12μL R6, spun 12,000RPM 30s

Quantified post-bisulfite-treated sample on NanoDrop1000:

(http://eagle.fish.washington.edu/Arabidopsis/20150227_Emma_1000ppm_pool_postBS_plot.JPG)

Definitely a low yield (~108ng) relative to the input (~400ng). Will proceed with Illumina library prep.

Library Prep

Illumina library prep was performed with [EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/Epigentek_PostBisulfiteIlluminaLibraryPrep_P-1055.pdf).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.

  • PCR cycles: 15

No other changes were made to the manufacturer’s protocol.

Epigentek Barcode Indices assigned, per their recommendations for using two libraries for multiplexing (this will be combined with the 400ppm library):

Barcode #12 – CTTGTA

The library was stored @ -20C and will be checked via Bioanalyzer on Monday.