alias: cDNA_Lib 13
center_name: BI
expected_number_runs: 3

STUDY_REF: G1828
SAMPLE_DESCRIPTOR: BioSam 1247

LIBRARY
LIBRARY_NAME: cDNA_Lib 13
LIBRARY_STRATEGY: mRNA-Seq
LIBRARY_SOURCE: TRANSCRIPTOMIC
LIBRARY_SELECTION: cDNA
LIBRARY_LAYOUT: SINGLE
POOLING_STRATEGY: multiplexed libraries
LIBRARY_CONSTRUCTION_PROTOCOL: Direct RNA-seq method based on RNA 3' linker ligation, unpublished

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: 101

BASE CALLS
GAPipeline RTA1.15.19.5

QUALITY SCORES
GAPipeline RTA1.15.19.5

ATTRIBUTES
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 3' cDNA ligation
RNA_PREPARATION_3'_RNA ADAPTER_LIGATION_PROTOCOL: Our protocol using T4 RNA ligase from NEB
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5ILL-21
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: ILLUMINA A1+B1, C1+D1, E1+F1, G1+H1
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: C3trunc
MRNA_PREPARATION_INITIAL_MRNA_QNTY: around 10ng
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 3x RLT + 15/20 ul of Silane + 0.4x EtOn, eluted in 100ul, recleaned using 0.95X SPRI
RNA_PREPARATION_5'_DEPHOSPHORYLATION: yes
EXTRACTION_PROTOCOL_MRNA_ENRICHMENT: OligodT - Dynabeads mRNA purification kit (Invitrogen)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: 98C 30 sec, 8 cycles of: 98C for 10 sec, 67C for 20 sec, 72C for 20 min, then 72C for 30 seconds, hold 4C
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: AR2
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: Phusion High Fidelity
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 8
LIBRARY_GENERATION_PCR_PRIMER_CONC: 12 pmoles of each/50 ul of reaction
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 2P_Universal
EXTRACTION_PROTOCOL_FRAGMENTATION: 70C for 4 min in Ambion Fragmentation Buffer
RNA_PREPARATION_5'_PHOSPHORYLATION: NO
EXPERIMENT_TYPE: mRNA-Seq
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: We synthesized first-strand cDNA using affinity-script reverse transcriptase
MRNA_PREPARATION_FRAGMENT_SIZE_RANGE: 50-300 nt