title: Maca transcriptome
design: The poly (A) mRNA isolation, fragmentation, and priming was performed using the NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB). First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase (NEB). Second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix (NEB). Double-stranded cDNA purified using AxyPrep Mag PCR Clean-up (Axygen) was then treated with End Prep Enzyme Mix (NEB) for end repair, 5” Phosphorylation, and dA-tailing in one reaction, followed by ligation to adaptors with a “T” base overhang. Size selection of Adaptor-ligated DNA was then performed using a AxyPrep Mag PCR Clean-up kit (Axygen), and fragments of ~400 bp (with the approximate insert size of 250 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences that can anneal with a flow cell to enable bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using an AxyPrep Mag PCR Clean-up kit (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies), and quantified via Qubit and real time PCR analysis (Applied Biosystems). Libraries with different indexes were then multiplexed and analyzed with an Illumina HiSeq 2500 instrument according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x125 paired-end (PE) configuration; image analysis and base calling were conducted with HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. The sequences were processed and analyzed with GENEWIZ software.