GSM3104653
GSM3104653: M1T1_WT-1; Streptococcus pyogenes; RNA-Seq
Total RNA was extracted using  Norgen RNA isolation kit (canada).The high quality total RNA samples were obtained by RNase-free DNase treatment. The samples were then sent to Novogene Co. The RNA purity, integrity and quantification were measured using Nanodrop, Qbit and agilent 2100.  After total RNA sample QC, Ribo-zero kits were used to remove rRNAs, then fragmentation buffer were added to fragment the mRNAs. Using random hexamers as primers, the mRNA fragments were reverse-transcribed to single stranded cDNAs. After the systhesis of single stranded cDNAs, buffer, dNTPs (in which dTTP were replaced by dUTP), DNA polymerase I and RNase H were applied to systhesis the complementary cDNA strands. The double stranded cDNAs were purified using AMPure XP beads. The double stranded cDNAs were end-repaired, polyadenylated, ligated with adapter squences and size-selected using AMPure XP beads. Then the uracil containing strands were degraded by USER Enzyme, and the remained strands were amplified using PCR and purified using AMPure XP beadsd. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/μl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM)..Library were sequenced using IlluminaHiseq 4000 HWI_ST1276 as instrument identifier and C1162ACXX as flow cell ID.
GEO
GSM3104653
GEO Accession
GSM3104653