alias: polyA-RNA-seq_h1-mesendoderm_r1a
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-Mesendoderm_RNA_r1

LIBRARY
LIBRARY_NAME: polyA-RNA-seq_h1-mesendoderm_r1a
LIBRARY_STRATEGY: mRNA-Seq
LIBRARY_SOURCE: TRANSCRIPTOMIC
LIBRARY_SELECTION: cDNA
LIBRARY_LAYOUT: PAIRED
POOLING_STRATEGY: none
LIBRARY_CONSTRUCTION_PROTOCOL: Four µg 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°C for 4 minutes. First-strand cDNA was synthesized with the following reaction composition: 0.5 µL random hexamer, 0.5 µL RNase Out, 1 µL DTT (100 mM), 0.5 µL dNTP (25 mM), 0.5 µL SuperScript III (20 µL final). The reaction was incubated at 25°C for 10 min then 50°C 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 µL NEBuffer 2 (NEB), 1 µL dNTP mix (10 mM dATP, dCTP, dGTP, and dUTP), 0.2 µL RNase H, 1 µL DNA Polymerase I (E. coli) (New England Biolabs) (15 µL final). The reaction was incubated at 16°C 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°C 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°C 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 µL TruSeq PCR Primer Cocktail (Illumina), 25 µL TruSeq PCR Master Mix (Illumina) (50 µL final). The thermocycling parameters were: 98°C 30 sec, then 10-15 cycles of 98°C 10 sec, 60°C 30 sec and 72°C 30 sec, ending with one 72°C 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 µg
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°C 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°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)
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°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 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/µL
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 200nM