GSM4631357: M_florum RNAseq - rep 1A; Mesoplasma florum; RNA-Seq
Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research, R2052) according to the manufacturer's specifications. RNA-Seq: ~300 ng of total RNA was fragmented at 95°C by Mg2+ catalyzed hydrolysis, and then purified using the RNA Clean & Concentrator-5 kit (Zymo Research). RNA molecules were dephosphorylated using Antarctic Phosphatase (NEB), and purified again using the RNA Clean & Concentrator-5 kit. The 3' sequencing adaptor was ligated using the T4 RNA ligase (Enzymatics), and RNA fragments were purified using Agencourt RNAclean XP SPRI beads (Beckman Coulter). RNA fragments were phosphorylated using T4 Polynucleotide Kinase (Enzymatics), purified again using Agencourt RNAclean XP SPRI beads, and the 5' sequencing adaptor was ligated using the T4 RNA ligase. RNA fragments were purified using Agencourt RNAclean XP SPRI beads, and then reverse transcribed to cDNA using the M-MuLV Reverse Transcriptase (Enzymatics). cDNA was purified using Agencourt AMPure XP SPRI beads, amplified by real-time PCR using the VeraSeq 2.0 DNA polymerase (Enzymatics), and purified again using Agencourt AMPure XP SPRI beads. DNA molecules corresponding to rRNA transcripts were depleted using the Duplex-specific nuclease (DSN, Evrogen). Final library size, quality, and concentration was evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies). RNA-Seq libraries were sequenced on a HiSeq 2000 Illumina instrument using 2x50 bp paired-end sequencing. For more details, see Carraro et al 2014. 5'-RACE: ~2 µg of total RNA was fragmented at 75°C by Mg2+ catalyzed hydrolysis, and then purified using the RNA Clean & Concentrator-5 kit (Zymo Research). The 3' sequencing adaptor was ligated using the T4 RNA ligase (Enzymatics), and RNA molecules were purified again using the RNA Clean & Concentrator-5 kit. Monophosphorylated RNA fragments were depleted using the XRN-1 nuclease (NEB), and the remaining RNA molecules were purified with the RNA Clean & Concentrator-5 kit. RNA fragments were treated with the RppH Pyrophosphohydrolase (NEB) to remove a pyrophosphate from the 5'-end of triphosphorylated RNA, and purified once again with the RNA Clean & Concentrator-5 kit. The 5' sequencing adaptor was finally ligated using the T4 RNA ligase, RNA fragments were purified using Agencourt RNAclean XP SPRI beads (Beckman Coulter), and reverse transcribed using the M-MuLV Reverse Transcriptase (Enzymatics). cDNA was purified using Agencourt AMPure XP SPRI beads, amplified by real-time PCR using the VeraSeq 2.0 DNA polymerase (Enzymatics), and purified again using Agencourt AMPure XP SPRI beads. Final library size, quality, and concentration was evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies). The 5'-RACE library was sequenced on an Illumina Genome Analyzer IIx instrument using 40 bp single-end sequencing. For more details, see Carraro et al 2014.
GEO Accession
GSM4631357