Instrument: Illumina Genome Analyzer IIx
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Total RNA was extracted using TRIzol® Reagent (Invitrogen), and RNA was precipitated by addition of 250 µL 100% isopropanol and 250 µL 1.2 M NaCitrate, 0.8 M NaCl in DEPC-treated water. The Zymo Research RNA Clean & Concentrator™-25 In-Column DNase Digestion protocol and 10 units Ambion® TURBO™ DNase was used on ~25 µg total RNA. Poly(A)+ transcripts were subsequently isolated from total RNA using Ambion® MicroPoly(A)Purist™ Kit. One hundred ng mRNA was chemically fragmented to a range of approximately 200-500 base pairs using the Ambion® RNA Fragmentation Reagent, and the reaction was cleaned using Zymo Research RNA Clean & Concentrator™-5. First strand cDNA was synthesized using SuperScript® II Reverse Transcriptase (Invitrogen™) and a combination of 3 µg random hexamers and 0.15 µg oligo(dT)20 primers. The second strand of the cDNA was synthesized using DNA polymerase I (Invitrogen™), RNase H (New England Biolabs® Inc.), dNTPs and second strand buffer (Invitrogen™). This reaction and all subsequent reactions were cleaned using Zymo Research DNA Clean & Concentrator™-5 kit. Double stranded cDNA templates were blunt ended using End-It™ Repair Kit (Epicentre®). A-overhangs were added to both ends with Klenow fragment (3'→5' exo-minus) (New England Biolabs® Inc.) and Illumina sequencing adapters were then ligated to both ends of the cDNA templates using Fast-Link™ DNA Ligation Kit (Epicentre®). cDNA templates were amplified by performing PCR for 18 cycles, which extended the adapter and incorporated a different six base pair index into each sample. The product was then isolated by gel purification of 250-550 base pair fragments.