alias: cDNA_Lib 2
center_name: BI
expected_number_runs: 2

STUDY_REF: G1828
SAMPLE_DESCRIPTOR: SRS255276

LIBRARY
LIBRARY_NAME: cDNA_Lib 2
LIBRARY_STRATEGY: mRNA-Seq
LIBRARY_SOURCE: TRANSCRIPTOMIC
LIBRARY_SELECTION: cDNA
LIBRARY_LAYOUT: SINGLE
POOLING_STRATEGY: multiplexed libraries
LIBRARY_CONSTRUCTION_PROTOCOL: Strand specific cDNA libraries were constructed for Illumina sequencing using the dUTP method essentially as described in S. Zhong et al, Cold Spring Harbor Protoc 8, 940 - 949 (2011).

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

PLATFORM
ILLUMINA
INSTRUMENT_MODEL: Illumina HiSeq 2000
SEQUENCE_LENGTH: 76

BASE CALLS
GAPipeline RTA1.12.4.2

QUALITY SCORES
GAPipeline RTA1.12.4.2

ATTRIBUTES
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: NA
RNA_PREPARATION_3'_RNA ADAPTER_LIGATION_PROTOCOL: NA
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: NA
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: NA
MRNA_PREPARATION_INITIAL_MRNA_QNTY: 200ng
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 1.8X SPRI cleanup (no size selection)
RNA_PREPARATION_5'_DEPHOSPHORYLATION: NA
EXTRACTION_PROTOCOL_MRNA_ENRICHMENT: Dynabeads mRNA purification kit (Invitrogen)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: 95C 2mins, 10 cycles of: 98C for 30 sec, 55C for 30 sec, 72C for 1 min, then 72C for 10 min, hold 4C
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: NNNNNN
EXTRACTION_PROTOCOL: Qiagen AllPrep DNA/RNA/Protein Mini Kit
LIBRARY_GENERATION_PCR_TEMPLATE: Adapter ligated cDNA made by reverse transcription of RNA
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: PFU Ultra II
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 10
LIBRARY_GENERATION_PCR_PRIMER_CONC: 0.83 uM
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: CAAGCAGAAGACGGCATACGAGAT
EXTRACTION_PROTOCOL_FRAGMENTATION: 98C for 40 min in 0.2 mM sodium citrate, pH 6.4 (Ambion), followed by concentrating it to 3.5 ul, mixing with 1 ul 2 uM SMART tagged random primer, incubating at 70deg C for 10 min and chilling on ice for 2 min.
RNA_PREPARATION_5'_PHOSPHORYLATION: NA
EXPERIMENT_TYPE: mRNA-Seq
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: We synthesized first-strand cDNA with this RNA primer mix by adding 4 ul 5x first-strand buffer, 2 ul 100 mM DTT, 1 ul 10 mM dNTPs, 4 ug of actinomycin D, 200 U SuperScript III and 20 U SUPERase-In, incubating at room temperature for 10 min followed by 1 h at 55degC. We cleaned up first-strand cDNA by PCIA extraction twice, ethanol precipitation with 0.1 volumes 5 M ammonium acetate to remove dNTPs and resuspension in 104 ul H2O. We synthesized second-strand cDNA by adding 4 ul of 5x first-strand buffer, 2 ul of 100 mM DTT, 4 ul of 10 mM dNTPs with dTTP replaced by dUTP (Sigma), 30 ul of 5x second-strand buffer, 40 U of Escherichia coli DNA polymerase, 10 U of E. coli DNA ligase and 2 U of E. coli RNase H, and incubating at 16degC for 2 h.
MRNA_PREPARATION_FRAGMENT_SIZE_RANGE: 400bp