Ricinus communis developing endosperm II/III transcriptome sequencing
mRNA-Seq of Ricinus communis developing endosperm II/III for digital transcriptomics
Transcriptome libraries were constructed using the Illumina mRNA Seq sample preparation kit with some modifications. The mRNA was enriched using the MicroPoly(A)Purist Kit with two rounds of oligo dT purification (Ambion). The mRNA quality was checked on a Nano chip of the Bioanalyser 2100 (Agilent) and the quantity measured with the Qubit RNA kit and Qubit fluorometer (Invitrogen).  In brief, Purified mRNA was fragmented by addition of 5x fragmentation buffer (Illumina, Hayward, CA) and was heated for 2 min at 94°C in a thermocycler. The shorter fragmentation time was used to yield library size of 350-400bp. First strand cDNA was synthesised using random primers to eliminate the general bias towards 3’ end of the transcript. Second strand cDNA synthesis was done by adding GEX second strand buffer (Illumina, Hayward, CA), dNTPs, RNaseH and DNA polymerase I followed by incubation for 2.5 h at 16°C. Second strand cDNA was further subjected to end repair, A-tailing, and adapter ligation in accordance with the manufacturer supplied protocols. Purified cDNA templates were enriched by 15 cycles of PCR for 10 s at 98°C, 30 s at 65°C, and 30 s at 72°C using PE1.0 and PE2.0 primers and with Phusion DNA polymerase (Illumina, Hayward, CA). The samples were cleaned using QIAquick PCR purification columns and eluted in 30 µl EB buffer as per manufacturer's instructions (QIAGEN, CA). Purified cDNA libraries were quantified using Bioanalyser DNA 100 Chip (Agilent).
TGAC
Developing_endosperm_II/III