alias: methylC-seq_h1+bmp4_r2c
expected_number_runs: 4
expected_number_spots: 60000000000
expected_number_reads: 40000000000
center_name: UCSD

STUDY_REF: The San Diego Epigenome Center
SAMPLE_DESCRIPTOR: H1+BMP4_r2

LIBRARY
LIBRARY_NAME: methylC-seq_h1+bmp4_r2c
LIBRARY_STRATEGY: Bisulfite-Seq
LIBRARY_SOURCE: GENOMIC
LIBRARY_SELECTION: RANDOM
LIBRARY_LAYOUT: SINGLE
LIBRARY_CONSTRUCTION_PROTOCOL: 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 16?C 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: 95?C 2 min, 98?C 30 sec, then 4-8 cycles of 98?C 15 sec, 60?C 30 sec and 72?C 4 min, ending with one 72?C 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 two independent libraries from the same biological sample.

SPOT
NUMBER_OF_READS_PER_SPOT: 1
READ_INDEX: 0
READ_CLASS: Application Read
READ_TYPE: Forward
BASE_COORD: 1

PLATFORM
ILLUMINA
INSTRUMENT_MODEL: Illumina Genome Analyzer II
CYCLE_SEQUENCE: normal
CYCLE_COUNT: 82

BASE CALLS
SEQUENCE_SPACE: Base Space
BASE_CALLER: Bustard

QUALITY SCORES
Quality Type: phred
QUALITY_SCORER: Bustard
NUMBER_OF_LEVELS: 1
MULTIPLIER: 1

ATTRIBUTES
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, 6 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: 16?C 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: >One third 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: 95?C 2 min; 98?C 30 sec, 4 cycles of 98?C 15 sec, 60?C 30 sec, 72?C 4 min; 72?C 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)