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polyA RNA sequencing of H1 cells
polyA RNA sequencing of H1 cells
Project News:
2012/3/30: EXPERIMENT XML file 'ecker.rna.h1.experiment.xml' uploaded
alias: polyA-RNA-seq_h1_r2a
expected_number_runs: 3 expected_number_spots: 30000000000 expected_number_reads: 20000000000 center_name: UCSD STUDY_REF: The San Diego Epigenome Center SAMPLE_DESCRIPTOR: H1_RNA_r2 LIBRARY LIBRARY_NAME: polyA-RNA-seq_h1_r2a LIBRARY_STRATEGY: mRNA-Seq LIBRARY_SOURCE: TRANSCRIPTOMIC LIBRARY_SELECTION: cDNA LIBRARY_LAYOUT: PAIRED POOLING_STRATEGY: none LIBRARY_CONSTRUCTION_PROTOCOL: Four 痢 of total RNA was extracted from frozen cell pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA). PolyA RNA was isolated from total RNA using Dynabeads oligo(dt)25 (Invitrogen). PolyA selected RNA was eluted in 2x Superscript III buffer supplemented with 10mM DTT and was fragmented by incubation at 94蚓 for 4 minutes. First-strand cDNA was synthesized with the following reaction composition: 0.5 無 random hexamer, 0.5 無 RNase Out, 1 無 DTT (100 mM), 0.5 無 dNTP (25 mM), 0.5 無 SuperScript III (20 無 final). The reaction was incubated at 25蚓 for 10 min then 50蚓 for 50 min. Single-stranded cDNA was isolated by purification with RNAClean XP beads (Beckman Coulter Genomics, Danvers, MA). The dUTP-containing second-strand was synthesized with the following reaction: 1.5 無 NEBuffer 2 (NEB), 1 無 dNTP mix (10 mM dATP, dCTP, dGTP, and dUTP), 0.2 無 RNase H, 1 無 DNA Polymerase I (E. coli) (New England Biolabs) (15 無 final). The reaction was incubated at 16蚓 for 2.5 hours. The dUTP-containing cDNA was isolated by purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). The dUTP-containing cDNA was then end repaired and a 3? A base was added. TruSeq adapters (Illumina, San Diego, CA) were ligated to the dUTP-containing cDNA at 16蚓 for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated cDNA was isolated by two rounds of purification with AMPure XP beads. The dUTP-containing strands were digested at 37蚓 for 30 min with uracil DNA glycosylase (Enzymatics). Adapter-ligated, single-stranded DNA molecules were enriched by 10 cycles of PCR with the following reaction composition: 5 無 TruSeq PCR Primer Cocktail (Illumina), 25 無 TruSeq PCR Master Mix (Illumina) (50 無 final). The thermocycling parameters were: 98蚓 30 sec, then 10-15 cycles of 98蚓 10 sec, 60蚓 30 sec and 72蚓 30 sec, ending with one 72蚓 5 min step. The reaction products were purified using AMPure XP beads. SPOT NUMBER_OF_READS_PER_SPOT: 2 READ_INDEX: 0 READ_CLASS: Application Read READ_TYPE: Forward BASE_COORD: 1 PLATFORM ILLUMINA INSTRUMENT_MODEL: Illumina HiSeq 2000 CYCLE_SEQUENCE: normal SEQUENCE_LENGTH: 100 BASE CALLS RTA 1.12.4.2 QUALITY SCORES RTA 1.12.4.2 ATTRIBUTES EXPERIMENT_TYPE: mRNA-Seq EXTRACTION_PROTOCOL: Qiagen RNeasy mini kit, performed as per manufacturer's instructions CDNA_PREPARATION_INITIAL_RNA_QNTY: 4 痢 CDNA_PREPARATION_POLYA_RNA/: Dynabeads oligo(dt)25 (Invitrogen) CDNA_PREPARATION_FRAGMENTATION: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94蚓 for 4 min CDNA_PREPARATION_FRAGMENT_SIZE_RANGE: 200-250 CDNA_PREPARATION_FIRST_STRAND_SYNTHESIS_ENZYME: SuperScript III (Invitrogen) CDNA_PREPARATION_FIRST_STRAND_PURIFICATION: Purification with RNAClean XP beads (Agencourt) CDNA_PREPARATION_SECOND_STRAND_SYNTHESIS_ENZYME: DNA Polymerase I (E. coli) (New England Biolabs) CDNA_PREPARATION_SECOND_STRAND_SYNTHESIS_DNTP_MIX: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP CDNA_PREPARATION_PURIFICATION: Purification with AMPure XP beads (Agencourt) DNA_PREPARATION_ADAPTOR: TruSeq Multiplexed Adapters (Illumina) DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL: 16蚓 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) DNA_PREPARATION_URACIL_DNA_GLYCOSYLASE_DIGESTION: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps. LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: TruSeq PCR Master Mix (Illumina) LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: 98蚓 30 sec; 10 cycles of 98蚓 10 sec, 60蚓 30 sec, 72蚓 30 sec; 72蚓 10 min LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 10 LIBRARY_GENERATION_PCR_PRIMER: TruSeq PCR Primer Cocktail (Illumina) LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: Purification with AMPure XP beads (Agencourt) EXTRACTION_PROTOCOL_MRNA_ENRICHMENT: Dynabeads oligo(dt)25 protocol RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: Random hexamers RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs) LIBRARY_GENERATION_PCR_TEMPLATE: cDNA LIBRARY_GENERATION_PCR_TEMPLATE_CONC: 5 ng/無 LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC 3' LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3' LIBRARY_GENERATION_PCR_PRIMER_CONC: 200nM |
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