polyA RNA sequencing of STL001 Bladder Cultured Cells
Four µg of total RNA was extracted from frozen cell pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA). PolyA RNA was isolated from total RNA using Dynabeads oligo(dt)25 (Invitrogen). PolyA selected RNA was eluted in 2x Superscript III buffer supplemented with 10mM DTT and was fragmented by incubation at 94°C for 4 minutes. First-strand cDNA was synthesized with the following reaction composition: 0.5 µL random hexamer, 0.5 µL RNase Out, 1 µL DTT (100 mM), 0.5 µL dNTP (25 mM), 0.5 µL SuperScript III (20 µL final). The reaction was incubated at 25°C for 10 min then 50°C for 50 min.  Single-stranded cDNA was isolated by purification with RNAClean XP beads (Beckman Coulter Genomics, Danvers, MA). The dUTP-containing second-strand was synthesized with the following reaction: 1.5 µL NEBuffer 2 (NEB), 1 µL dNTP mix (10 mM dATP, dCTP, dGTP, and dUTP), 0.2 µL RNase H, 1 µL DNA Polymerase I (E. coli) (New England Biolabs) (15 µL final). The reaction was incubated at 16°C for 2.5 hours. The dUTP-containing cDNA was isolated by purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). The dUTP-containing cDNA was then end repaired and a 3’ A base was added. TruSeq adapters (Illumina, San Diego, CA) were ligated to the dUTP-containing cDNA at 16°C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated cDNA was isolated by two rounds of purification with AMPure XP beads. The dUTP-containing strands were digested at 37°C for 30 min with uracil DNA glycosylase (Enzymatics). Adapter-ligated, single-stranded DNA molecules were enriched by 10 cycles of PCR with the following reaction composition: 5 µL TruSeq PCR Primer Cocktail (Illumina), 25 µL TruSeq PCR Master Mix (Illumina) (50 µL final). The thermocycling parameters were: 98°C 30 sec, then 10-15 cycles of 98°C 10 sec, 60°C 30 sec and 72°C 30 sec, ending with one 72°C 5 min step. The reaction products were purified using AMPure XP beads. 
EXPERIMENT_TYPE
mRNA-Seq
EXTRACTION_PROTOCOL
Qiagen RNeasy mini kit, performed as per manufacturer's instructions
CDNA_PREPARATION_INITIAL_RNA_QNTY
4 µg
CDNA_PREPARATION_POLYA_RNA/
Dynabeads oligo(dt)25 (Invitrogen)
CDNA_PREPARATION_FRAGMENTATION
PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min
CDNA_PREPARATION_FRAGMENT_SIZE_RANGE
200-250
CDNA_PREPARATION_FIRST_STRAND_SYNTHESIS_ENZYME
SuperScript III (Invitrogen)
CDNA_PREPARATION_FIRST_STRAND_PURIFICATION
Purification with RNAClean XP beads (Agencourt)
CDNA_PREPARATION_SECOND_STRAND_SYNTHESIS_ENZYME
DNA Polymerase I (E. coli) (New England Biolabs)
CDNA_PREPARATION_SECOND_STRAND_SYNTHESIS_DNTP_MIX
dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP
CDNA_PREPARATION_PURIFICATION
Purification with AMPure XP beads (Agencourt)
DNA_PREPARATION_ADAPTOR
TruSeq Multiplexed Adapters (Illumina)
DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
16°C for 16 hours with T4 DNA ligase (New England Biolabs)
DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
Two rounds of purification with AMPure XP beads (Agencourt)
DNA_PREPARATION_URACIL_DNA_GLYCOSYLASE_DIGESTION
One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.
LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
TruSeq PCR Master Mix (Illumina)
LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min
LIBRARY_GENERATION_PCR_NUMBER_CYCLES
10
LIBRARY_GENERATION_PCR_PRIMER
TruSeq PCR Primer Cocktail (Illumina)
LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
Purification with AMPure XP beads (Agencourt)
EXTRACTION_PROTOCOL_MRNA_ENRICHMENT
Dynabeads oligo(dt)25 protocol
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE
Random hexamers
RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL
First-strand Superscript III (Invitrogen) RT protocol. RNAClean XP purification of first strand product. Second-strand synthesis using dUTP-containing dNTPS and DNA Polymerase 1 (E. coli) (New England Biolabs)
LIBRARY_GENERATION_PCR_TEMPLATE
cDNA
LIBRARY_GENERATION_PCR_TEMPLATE_CONC
5 ng/µL
LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC
200nM
GEO Accession
GSM1067793