| Library |
| Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
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Total RNA was isolated from mixed organs of CM104, which included whole plant organs from seed germination to the two-leaf stage, plant shoots in the seedling stage, and leaves, stems, ears and seeds from the heading to late filling stage. The RNA was isolated according to the instructions for the TRIzol method. The integrity, quality and concentration of RNA were detected by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies). High-quality RNA (2 μg, 300 ng/μl) was prepared for the next step of library construction. PacBio SMRT bell library preparation was performed using a SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, PN 101-853-100) in accordance with the manufacturer's instructions (Procedure & Checklist - Iso-Seq Express Template Preparation for Sequel and Sequel II Systems). In brief, (i) first-strand cDNA was synthesized by reverse transcription with oligo (dT) primer to pair with the poly(A) tails. (ii) Template switching was performed by adding the other primer paired with TS oligo. (iii) Full-length cDNA was obtained by cDNA amplification with barcoded cDNA PCR primers. (iv) Amplified cDNA was purified with ProNex Beads and quantified by a Qubit fluorometer (Invitrogen). (v) The SMRTbell library was prepared using Kit 2.0. (vi) Single-strand overhangs were removed, and then we performed DNA damage repair, end repair, A-tailing and adapter ligation. After library purification using 0.45X AMPure PB Beads and 1-10 kb size selection, the library was prepared for sequencing with a 30 h movie on the Sequel IIe system (Pacific Biosciences, CA, USA) at the Berry Genomics Corporation (Beijing, China). |
ISO-Seq |
TRANSCRIPTOMIC |
PolyA |
SINGLE
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