miRNA-Seq analysis of brain (insular) tissue from NB141 (m09677)
miRNA-Seq analysis of brain (insular) tissue from NB141 (m09677) using Illumina HiSeq 2000
Refer to document 'miRNA3 - Plate Format miRNA Library Construction' from BC at the Roadmap Epigenomics Project site, Experimental Protocols page (URL: http://www.roadmapepigenomics.org/protocols/type/experimental/)
EXPERIMENT_TYPE
smRNA-Seq
EXTRACTION_PROTOCOL
Total RNA was extracted using Trizol from Invitrogen as per manufacturer's instructions.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
Phusion DNA Polymerase (Hot Start), 2U/ul (from NEB)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
step1: 98C for 30sec, step2: 98C for 10sec, step3: 60C for 30sec, step4: 72C for 15sec.  Repeat step2-4 for 15 cycles, 72C for 5min.
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
15
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
5' AATGATACGGCGACCACCGACAGNNNNNNGTTCAGAGTTCTACAGTCCGA 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC
25 uM
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.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT
Flowthrough of polyA+ MultiMACS 96 Separation Unit
SMRNA_PREPARATION_INITIAL_SMRNA_QLTY
RQS 6.2
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY
1.3262 ug
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 70C 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 22C in a Tetrad thermocycler for 60min.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL
5' RNA adapter was heat denatured at 70C 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 37C 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 65C 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 44C in a Thetrad thermocycler for 60 minutes.
LIBRARY_GENERATION_PCR_TEMPLATE
15ul of the first strand cDNA product was used in the PCR