alias: smRNA-seq_h1_r1
expected_number_runs: 1
expected_number_spots: 15000000
expected_number_reads: 10000000
center_name: UCSD

STUDY_REF: The San Diego Epigenome Center
SAMPLE_DESCRIPTOR: H1_r1

LIBRARY
LIBRARY_NAME: smRNA-seq_h1_r1
LIBRARY_STRATEGY: smRNA-Seq
LIBRARY_SOURCE: RNA
LIBRARY_SELECTION: cDNA
LIBRARY_LAYOUT: SINGLE
LIBRARY_CONSTRUCTION_PROTOCOL: The small RNA fraction was isolated from H1 cells then sequentially ligated to the adenylated 3' RNA adapter then to the 5' RNA adapter. Following reverse transcription of the adapter ligated small RNAs, the library was enriched by 12 cycles of PCR.

SPOT
SPOT_DECODE_SPEC: 1 0 Application Read Forward 1

PLATFORM
ILLUMINA
INSTRUMENT_MODEL: Illumina Genome Analyzer II
CYCLE_SEQUENCE: normal
CYCLE_COUNT: 43

BASE CALLS
SEQUENCE_SPACE: Base Space
BASE_CALLER: Bustard

QUALITY SCORES
Quality Type: phred
QUALITY_SCORER: Bustard
NUMBER_OF_LEVELS: 1
MULTIPLIER: 1

ATTRIBUTES
EXPERIMENT_TYPE: smRNA-Seq
EXTRACTION_PROTOCOL: mirVana miRNA isolation kit (Applied Biosystems), performed as per manufacturer's instructions to isolate small RNAs, followed by treatment with DNaseI (Qiagen) for 30 min at room temperature.
EXTRACTION_PROTOCOL_SMRNA_ENRICHMENT: The small RNA fraction were separated by electrophoresis on a 15% TBE-urea gel and RNA molecules between approximately 10 and 50 nt were excised and eluted from the gel fragments then ethanol precipitated, resuspending in 6 µl.
SMRNA_PREPARATION_INITIAL_SMRNA_QNTY: Equivalent to the quantity of smRNAs isolated from 5 µg of total RNA.
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE: 5' GUUCAGAGUUCUACAGUCCGACGAUC
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE: 5' UCGUAUGCCGUCUUCUGCUUGidT, 5' adenylated (Illumina)
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGA
RNA_PREPARATION_3'_RNA ADAPTER_LIGATION_PROTOCOL: 1 µl of 1:10 diluted adenylated 3? RNA adapter oligonucleotide was added to the 6 µl of smRNA and incubated at 70?C for 2 min followed by placement on ice. The 3? RNA adapter ligation reaction was performed by addition of 2 µl 5x T4 RNA ligase 2 truncated ligation buffer, 20 U RNaseOut and 300 U T4 RNA ligase 2 truncated (New England Biolabs) and incubation at 22?C for 1 h.
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL: Ligation of the 5? RNA adapter was performed by addition to the 3? adapter ligated reaction of 0.5 µl heat denatured (70?C 2 min) 5? RNA adapter oligonucleotide, 1 µl 10 mM ATP, and 10 U T4 RNA ligase (Promega), and incubation at 20?C for 6 h.
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL: To 4 µl of the RNA ligation products, 1 µl 1:5 diluted RT primer was added and heat denatured (70?C 2 min), followed by incubation on ice. Added to the denatured RNA/primer solution was 2 µl 5x first strand buffer, 0.5 µl 12.5 mM dNTPs, 1 µl 100 mM DTT, and 20 U RNaseOut, followed by incubation at 48?C for 3 min. To this, 100 U Superscript II reverse transcriptase (Life Technologies) was added, followed by incubation at 44?C for 1 h.
LIBRARY_GENERATION_PCR_TEMPLATE: The entire reverse transcription reaction was used in the PCR enrichment of the library.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE: Phusion hot-start high fidelity DNA polymerase (New England Biolabs). 1x Phusion polymerase buffer and 2 U Phusion hot-start high fidelity DNA polymerase was used in a 50 µl total reaction.
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM: 98?C 30 sec; 98?C 10 sec, 60?C 30 sec, 72?C 15 sec; 72?C 10 min
LIBRARY_GENERATION_PCR_NUMBER_CYCLES: 12
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE: 5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGA
LIBRARY_GENERATION_PCR_PRIMER_CONC: 0.25 µM
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL: PCR products were separated by electrophoresis on a 6% polyacrylamide gel and the PCR products (~90-125 bp) were excised, eluted from the crushed gel by rotation in 100 µl 1x gel elution buffer for 2 h, then ethanol precipitation of the DNA within the supernatant.