An improved repertoire of splicing variants and their potential roles in Arabidopsis photomorphogenic development

Total RNA was extracted from 4-d-old etiolated seedlings illuminated with 100 micro mole m-2s-1 white light for 4 h (L4h) or remained in the dark for 4 h (D4h) by using Plant RNA reagent (Invitrogen) and normalization of D4h and L4h cDNA libraries involved using the Trimmer-2 cDNA normalization kit (Evrogen). SMARTbell libraries of 1-2 and 2-4 kb cDNAs were prepared separately by pooling fractions 9 and 10 at a ratio of 7:3 (1-2 kb, 500 ng for library I) and fractions 6, 7 and 8 at ratio 1:3:6 (2-4 kb, 1 micro g for library II), according to size distribution of expressed genes.SMRTbell libraries, two each for libraries I and II, were prepared by using DNA Template Prep Kit 2.0 (Pacific Biosciences). SMRTbell libraries were prepared for sequencing by using the DNA polymerase binding kit P6 v2 primers (Pacific Biosciences) and the Magbead Binding Kit (Pacific Biosciences). The sequencing was performed on a PacBio RS II sequencer platform with eight v3 SMRTcells (Pacific Biosciences). Each of the four libraries was sequenced on two cells by C4 sequencing reagent (Pacific Biosciences) with 240-min signal collections.For non-normalized libraries, RNAs were isolated from 4-d-old etiolated seedlings (D4h) or 4-d-old etiolated seedling treated with 4-h light (L4h). PacBio libraries were prepared according to the Pacific Biosciences's protocol using the SMARTer PCR cDNA Synthesis kit (Clontech) and amplification by the KAPA DNA polymerase. PacBio SMRT cDNA libraries were prepared with the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) and sequenced on the PacBio Sequel I with Sequel DNA polymerase and binding kit and sequencing chemistry version 2.1 for 20 h on individual SMRT cell.