Whole genome shotgun bisulfite sequencing of the H9 cell line
Five µg of genomic DNA was extracted from frozen cell pellets using the DNeasy Mini Kit (Qiagen, Valencia, CA) and spiked with 25 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 100-150 bp, followed by end repair and addition of a 3' A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16degC for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (~450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer's instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 4 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5 µl 10X PfuTurbo reaction buffer, 31 µM dNTPs, 1 µl Primer 1, 1 µl Primer 2 (50 µl final). The thermocycling parameters were: 95degC 2 min, 98degC 30 sec, then 4-8 cycles of 98degC 15 sec, 60degC 30 sec and 72degC 4 min, ending with one 72degC 10 min step. The reaction products were purified using AMPure XP beads (two rounds). Up to three separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to three independent libraries from the same biological sample.
EXPERIMENT_TYPE
DNA Methylation
EXTRACTION_PROTOCOL
Qiagen DNeasy mini kit, performed as per manufacturer's instructions
EXTRACTION_PROTOCOL_TYPE_OF_SONICATOR
Covaris S2
EXTRACTION_PROTOCOL_SONICATION_CYCLES
Standard fragment express, 4 cycles
DNA_PREPARATION_INITIAL_DNA_QNTY
5 µg
DNA_PREPARATION_FRAGMENT_SIZE_RANGE
100-150
DNA_PREPARATION_ADAPTOR_SEQUENCE
A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
16degC for 16 hours with T4 DNA ligase (New England Biolabs)
DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
Two rounds of purification with AMPure XP beads (Agencourt)
BISULFITE_CONVERSION_PROTOCOL
Invitrogen MethylCode
BISULFITE_CONVERSION_PERCENT
99.5% of cytosines converted based on shotgun sequencing of unmethylated lambda phage control spiked into original genomic DNA sample
LIBRARY_GENERATION_PCR_TEMPLATE_CONC
>30-100% of the adapter-ligated, bisulfite converted DNA was used in each of three 50 µl PCR reaction
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
Stratagene Pfu Turbo Cx
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
95degC 2 min; 98degC 30 sec, 4 cycles of 98degC 15 sec, 60degC 30 sec, 72degC 4 min; 72degC 10 min
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
4
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
LIBRARY_GENERATION_PCR_PRIMER_CONC
25 µM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
Two rounds of purification with AMPure XP beads (Agencourt)
GEO Accession
GSM706060