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Whole genome shotgun bisulfite sequencing of the H1 cell line differentiated into neural progenitor cells
Whole genome shotgun bisulfite sequencing of the H1 cell line differentiated into neural progenitor cells
Project News:
2011/2/6: EXPERIMENT XML file 'ecker.methylC-seq_h1-npc.experiment.xml' uploaded
alias: methylC-seq_h1-npc_r2a
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-NPC_r2 LIBRARY LIBRARY_NAME: methylC-seq_h1-npc_r2a LIBRARY_STRATEGY: Bisulfite-Seq LIBRARY_SOURCE: GENOMIC LIBRARY_SELECTION: RANDOM LIBRARY_LAYOUT: SINGLE POOLING_STRATEGY: none 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 three 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 SEQUENCE_LENGTH: 86 BASE CALLS Bustard 1.8.70.0 QUALITY SCORES Bustard 1.8.70.0 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) |
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