ExperimentID:  Strand-specific, shotgun sequencing of mRNA from the IMR90 cell line||Mon Aug 10 14:59:09 -0500 2009||72554
LIBRARY_GENERATION_PCR_NUMBER_CYCLES:
15
LIBRARY_STRATEGY:
mRNA-Seq
LIBRARY_GENERATION_PCR_PRIMER_CONC:
0.25 µM
READ_INDEX:
0
LIBRARY_CONSTRUCTION_PROTOCOL:
Total RNA was isolated from IMR90 cells and mRNA isolated by RiboMinus depletion of rRNA. mRNA was fragmented, dephosphorylated, 5' phosphorylated, then sequentially ligated to the adenylated 3' RNA adapter then to the 5' RNA adapter. Following reverse transcription of the adapter ligated RNA, the library was enriched by 15 cycles of PCR.
READ_TYPE:
Forward
LIBRARY_LAYOUT:
SINGLE
expected_number_runs:
2
EXTRACTION_PROTOCOL:
mirVana miRNA isolation kit (Applied Biosystems), performed as per manufacturer's instructions to isolate total RNA, followed by treatment with DNaseI (Qiagen) for 30 min at room temperature
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM:
98ËšC 30 sec; 98ËšC 10 sec, 60ËšC 30 sec, 72ËšC 15 sec; 72ËšC 10 min
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL:
PCR products were purified in two steps, first by purification using 180 µl SPRI beads and elution in 30 µl 10 mM Tris buffer pH 8.0, followed by purification with 39 µl SPRI beads and elution in 10 µl 10 mM Tris buffer pH 8.0.
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE:
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA
READ_CLASS:
Application Read
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE:
Phusion hot-start high fidelity DNA polymerase (New England Biolabs). 1x Phusion polymerase buffer and 4 U Phusion hot-start high fidelity DNA polymerase was used in a 100 µl reaction.
LIBRARY_SOURCE:
cDNA
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE:
5' CAAGCAGAAGACGGCATACGA
BASE_COORD:
1
PLATFORM:
ILLUMINA
SEQUENCE_SPACE:
Base Space
QUALITY_TYPE:
phred
NUMBER_OF_READS_PER_SPOT:
1
MRNA_PREPARATION_INITIAL_MRNA_QNTY:
200 ng
RNA_PREPARATION_3'_RNA_ADAPTER_SEQUENCE:
5' UCGUAUGCCGUCUUCUGCUUGidT, 5' adenylated (Illumina)
MRNA_PREPARATION_FRAGMENT_SIZE_RANGE:
50-400 bp
LIBRARY_GENERATION_PCR_TEMPLATE:
The entire reverse transcription reaction was used in the PCR enrichment of the library.
RNA_PREPARATION_5'_RNA_ADAPTER_SEQUENCE:
5' GUUCAGAGUUCUACAGUCCGACGAUC
EXTRACTION_PROTOCOL_FRAGMENTATION:
15 min at 70 ËšC with RNA Fragmentation kit (Ambion/Applied Biosystems) as per manufacturer's instructions
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE:
5' CAAGCAGAAGACGGCATACGA
RNA_PREPARATION_5'_RNA_ADAPTER_LIGATION_PROTOCOL:
Ligation of the 5’ RNA adapter was performed by addition to the 3’ adapter ligated reaction of 1 µl 1:1 diluted, 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 1 h. RNA was purified using 66 µl SPRI beads and eluted in 10 µl 10 mM Tris buffer pH 8.0.
RNA_PREPARATION_5'_PHOSPHORYLATION:
Phosphorylation was performed by addition of 10 U PNK (New England Biolabs), 1 mM ATP, and 20 U RNaseOut  (Life Technologies) and incubation at 37ËšC for 1 h.
EXTRACTION_PROTOCOL_MRNA_ENRICHMENT:
2 x RiboMinus (Life Technologies) rRNA depletion (5S, 5.8S, 12S, 18S, 28S)
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL:
To the RNA ligation products, 2 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 4 µl 5x first strand buffer, 1 µl 12.5 mM dNTPs, 2 µl 100 mM DTT, and 40 U RNaseOut, followed by incubation at 48˚C for 1 min. To this, 200 U Superscript II reverse transcriptase (Life Technologies) was added, followed by incubation at 44˚C for 1 h.
RNA_PREPARATION_5'_DEPHOSPHORYLATION:
Fragmented RNA was treated with 5 U Antarctic phosphatase (New England Biolabs) for 40 min at 37˚C in the presence of 40 U RNaseOut followed by phosphatase heat inactivation at 65˚C for 5 min. The RNA was purified using 66 µl SPRI beads (Agencourt) and eluted in 11 µl 10 mM Tris buffer pH 8.0.