title: polyA RNA sequencing of H1 derived mesenchymal cells
design: 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. 
attributes.tag: EXPERIMENT_TYPE
attributes.value: mRNA-Seq
attributes.tag: EXTRACTION_PROTOCOL
attributes.value: Qiagen RNeasy mini kit, performed as per manufacturer's instructions
attributes.tag: CDNA_PREPARATION_INITIAL_RNA_QNTY
attributes.value: 4 µg
attributes.tag: CDNA_PREPARATION_POLYA_RNA/
attributes.value: Dynabeads oligo(dt)25 (Invitrogen)
attributes.tag: CDNA_PREPARATION_FRAGMENTATION
attributes.value: PolyA RNA resuspended in 2x Superscript III buffer supplemented with 10 mM DTT was incubated at 94°C for 4 min
attributes.tag: CDNA_PREPARATION_FRAGMENT_SIZE_RANGE
attributes.value: 200-250
attributes.tag: CDNA_PREPARATION_FIRST_STRAND_SYNTHESIS_ENZYME
attributes.value: SuperScript III (Invitrogen)
attributes.tag: CDNA_PREPARATION_FIRST_STRAND_PURIFICATION
attributes.value: Purification with RNAClean XP beads (Agencourt)
attributes.tag: CDNA_PREPARATION_SECOND_STRAND_SYNTHESIS_ENZYME
attributes.value: DNA Polymerase I (E. coli) (New England Biolabs)
attributes.tag: CDNA_PREPARATION_SECOND_STRAND_SYNTHESIS_DNTP_MIX
attributes.value: dNTP mix made from 10 mM dATP, dCTP, dGTP, dUTP
attributes.tag: CDNA_PREPARATION_PURIFICATION
attributes.value: Purification with AMPure XP beads (Agencourt)
attributes.tag: DNA_PREPARATION_ADAPTOR
attributes.value: TruSeq Multiplexed Adapters (Illumina)
attributes.tag: DNA_PREPARATION_ADAPTOR_LIGATION_PROTOCOL
attributes.value: 16°C for 16 hours with T4 DNA ligase (New England Biolabs)
attributes.tag: DNA_PREPARATION_POST-LIGATION_FRAGMENT_SIZE_SELECTION
attributes.value: Two rounds of purification with AMPure XP beads (Agencourt)
attributes.tag: DNA_PREPARATION_URACIL_DNA_GLYCOSYLASE_DIGESTION
attributes.value: One half of adapter-ligated cDNA digested with uracil DNA glycosylase (Enzymatics) and used for subsequent steps.
attributes.tag: LIBRARY_GENERATION_PCR_POLYMERASE_TYPE
attributes.value: TruSeq PCR Master Mix (Illumina)
attributes.tag: LIBRARY_GENERATION_PCR_THERMOCYCLING_PROGRAM
attributes.value: 98°C 30 sec; 10 cycles of 98°C 10 sec, 60°C 30 sec, 72°C 30 sec; 72°C 10 min
attributes.tag: LIBRARY_GENERATION_PCR_NUMBER_CYCLES
attributes.value: 10
attributes.tag: LIBRARY_GENERATION_PCR_PRIMER
attributes.value: TruSeq PCR Primer Cocktail (Illumina)
attributes.tag: LIBRARY_GENERATION_PCR_PRODUCT_ISOLATION_PROTOCOL
attributes.value: Purification with AMPure XP beads (Agencourt)
attributes.tag: EXTRACTION_PROTOCOL_MRNA_ENRICHMENT
attributes.value: Dynabeads oligo(dt)25 protocol
attributes.tag: RNA_PREPARATION_REVERSE_TRANSCRIPTION_PRIMER_SEQUENCE
attributes.value: Random hexamers
attributes.tag: RNA_PREPARATION_REVERSE_TRANSCRIPTION_PROTOCOL
attributes.value: 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)
attributes.tag: LIBRARY_GENERATION_PCR_TEMPLATE
attributes.value: cDNA
attributes.tag: LIBRARY_GENERATION_PCR_TEMPLATE_CONC
attributes.value: 5 ng/µL
attributes.tag: LIBRARY_GENERATION_PCR_F_PRIMER_SEQUENCE
attributes.value: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC 3'
attributes.tag: LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE
attributes.value: 5' CAAGCAGAAGACGGCATACGAGAT 3'
attributes.tag: LIBRARY_GENERATION_PCR_PRIMER_CONC
attributes.value: 200nM
attributes.tag: GEO Accession
attributes.value: GSM915323