title: GSM2262558: KPC; Mus musculus; RNA-Seq
library_construction_protocol: Samples' total RNA was extracted by Trizol and 200ng total RNA sample was purified by oligo-dT beads. mRNA isolation and fragmentation: 200ng total RNA sample was purified by oligo-dT beads, then poly (A)-containing mRNA were fragmented into small pieces with Elute, Prime, Fragment Mix . cDNA synthesis: First-strand cDNA was generated by First Strand Master Mix and Super Script II(Invitrogen) reverse transcription(Reaction condition：25℃ for 10 min；42℃ for 50 min；70℃for 15 min). Then adding Second Strand Master Mix to synthes the second-strand cDNA (16℃1h). End repair, add A and adaptor ligation: the purified Fragmented cDNA combine with End Repair Mix, incubate at30℃ 30min. Purify the end-repaired DNA with Ampure XP Beads (AGENCOURT). Then add A-Tailing Mix , mix well by pipetting, incubate at 37℃ 30 min. Adapter Ligation : Combine the Adenylate 3'Ends DNA, RNA Index Adapter and Ligation Mix, mix well by pipetting. Incubate the ligate reaction at 30℃ 10 min. Purify the end-repaired DNA with Ampure XP Beads (AGENCOURT). RCR: Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments. Then the PCR products are purified with Ampure XP Beads (AGENCOURT).
attributes.tag: GEO Accession
attributes.value: GSM2262558