EXPERIMENT_TYPE: mRNA-Seq
EXTRACTION_PROTOCOL:
Qiagen RNeasy mini kit, performed as per manufactu (show full text...)(hide...)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 (show full text...)(hide...)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 (show full text...)(hide...)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 (A (show full text...)(hide...)Two rounds of purification with AMPure XP beads (Agencourt)
DNA_PREPARATION_URACIL_DNA_GLYCOSYLASE_DIGESTION:
One half of adapter-ligated cDNA digested with ura (show full text...)(hide...)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 (show full text...)(hide...)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 proto (show full text...)(hide...)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' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC (show full text...)(hide...)5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC 3'
LIBRARY_GENERATION_PCR_R_PRIMER_SEQUENCE: 5' CAAGCAGAAGACGGCATACGAGAT 3'
LIBRARY_GENERATION_PCR_PRIMER_CONC: 200nM
GEO Accession: GSM1010942