GSM1214807: WT_36oC_15min; Schizosaccharomyces pombe; RNA-Seq
Qiagen RNeasy Mini Kit was used to extract total RNA. 1. Poly-A RNA is purified from total RNA (100 ng to 4000 ng) using poly-T oligo-attached magnetic beads.   2. Purified poly-A RNA is eluted from the magnetic beads and is simultaneously fragmented.  Cleaved RNA fragments are primed with random hexamers.  3. Randomly primed RNA fragments are used to generate first strand cDNA using reverse transcriptase.  4. The RNA template strand is removed from the first strand cDNA and the second strand cDNA is synthesized.  5. Ampure XP beads are used to purify the ds cDNA from the second strand reaction mix.  6.   Overhangs on the double-stranded cDNA are removed using an End Repair mix which results in the generation of blunt ends.  A 3' to 5' exonuclease activity in this mix removes the 3' overhangs and a polymerase activity fills in the 5' overhangs.  7. The blunt-ended cDNA fragments are purified using Ampure XP Beads.  8. A single  %22A%22 nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction.    9. Indexed adapters are ligated to the ends of the ds cDNA. A corresponding  %22T%22 residue on the 3' end of the adapter provides a complimentary overhang for ligating the adapter to the %22A%22 overhang on the cDNA fragment.  These adapters include all sequences needed for hybridization to a sequencing flowcell .   10. Adapter-ligated cDNA molecules are purified with Ampure XP Beads.  11. PCR is performed to enrich those DNA fragments that have adapter molecules ligated to both ends.  The PCR reaction is performed using primers that anneal to the ends of the adapters.  A total of 15 cycles of PCR is performed.  12. PCR amplified library molecules are purified using Ampure XP beads.  13. The concentration of the amplified library is measured on a NanoDrop spectrophotometer.  14. An aliquot of the amplified library is run on a DNA1000 bioanalyzer chip to validate the approximate size range of the library.  15. The concentration of cluster forming units in the library is defined by qPCR using the Kapbiosystems KAPA Library Quant Kit.
GEO
GSM1214807