EXPERIMENT XML file 'experiment.REMC_20110517_miRNA_Seq.xml' uploaded
LIBRARY_CONSTRUCTION_PROTOCOL: LIBPR.0054 miRNA3 - Plate format miRNA Library Construction
READ_CLASS: Application Read
INSTRUMENT_MODEL: Illumina Genome Analyzer II
Illumina RTA 126.96.36.199
Illumina RTA 188.8.131.52
EXTRACTION_PROTOCOL: Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instuctions.
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY: RIN 7.1
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GTTCAGAGTTCTACAGTCCGACGATCTGGTCAA 3'
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' ATCTCGTATGCCGTCTTCTGCTTGT 3'
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
RNA_PREPARATION_3'_RNA_ADAPTER_LIGATION_PROTOCOL: 1ug of total RNA (RN=>7.0) in a 4uL volume and 2uL of 3' Adenylated Adapter was heat denatured at 70°C for 2min then snap chilled on ice for 1min. Then 1uL of 10X T4 RNL2 truncated buffer, 0.8uL 100mM MgCl2, 0.2uL DEPC water, 0.5uL Rnase Out, and 1.5uL T4 RNA Ligase 2 trancated was added. Ligation incubated at 22°C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: 5' RNA adapter was heat denatured at 70°C for 2min then snap chilled on ice. 2uL of denatured 5' RNA Adapter was added to the 3' DNA Adapter Ligation reaction product from previous step and mixed. Then 1uL of 10mM ATP and 1uL of Ambion T4 RNA Ligase were added and ligation reaction was incubated at 37°C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To the 14uL of double adaptered miRNA product from previous step 2ul of RT-Primer was added and the mixture was heat denatured at 65°C for 10 mins then quenched on ice. The following reagents were added immediately to the sample (6ul of 5X First Strand Buffer, 2ul of 10mM dNTPs , 3ul of 100mM DTT, 1ul of RNaseOUT, and 2uL of SuperScript II RT. Reaction was heated at 44°C in a Thetrad thermocycler for 60minutes.
LIBRARY_GENERATION_PCR_TEMPLATE: 15ul of the first strand cDNA product was used in the PCR
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: step1: 98°C for 30sec, step2: 98°C for 10sec, step3: 60°C for 30sec, step4: 72°C for 15sec. Repeat step2-4 for 15 cycles, 72°C for 5min.
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: 28uL of PCR products containing unique index sequences are pooled together and the 115bp miRNA containing fraction is isolated for each pool either manually: 12% PAGE gel 200V 6hours followed by gel elution, or robotically using Baraccuda size selection robot. Fractions are Et0H preciptated, QCed on Agilent, and submitted for indexing single end (ISE) Illumina sequencing.