Iso seq express oligo kit

RNA was isolated and quantified as for short read sequencing above. Samples with RIN ≥ 7 were used to prepare the IsoSeq barcoded libraries following the PacBio protocol (Procedure & Checklist–Iso-SeqExpress Template Preparation for Sequel and Sequel II Systems, PN 101-763-800 Version 02) using the SMRTbell Express Template Prep kit 2.0 (PacBio). The individual libraries were barcoded using the Iso-Seq Express Oligo kit (PacBio PN 101-737-500). The barcoded libraries were multiplexed in equimolar amounts into a final SMRTbell template, which was purified using 1X beads. The final library was sequenced on the Sequel II system for 20 hours using the Sequel II Binding kit 2.1. The final loading concentration was 80 pM.

For whole genome sequencing, a SMRTbell library with an insert size of 12,000 nt was prepared from the high molecular weight DNA template using SMRTbell Express Template Prep Kit 2.0 and sequenced on the PacBio Sequel system (Pacific Biosciences, Menlo Park, CA, USA). Sequencing was performed with the Sequel Binding Kit 2.0 using a 20-h movie collection time following the manufacturer’s protocol (Pacific Biosciences, Menlo Park, CA, USA). For transcriptome sequencing, Iso-seq libraries were prepared using the NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs, Ipswich, MA, USA), Iso-Seq Express Oligo Kit, and SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). Iso-seq was performed as described above.

The male and female samples from D. magna were prepared for long-read isoform sequencing (Iso-Seq). Three biological replicates for each sample were conducted. The full-length cDNA was produced from 300 ng of total RNA for each sample using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England Biolabs, MA USA) and Iso-Seq Express Oligo kit (Pacific Biosciences, CA USA) according to manufacturer’s protocols. Additionally, ribosomal RNA (rRNA) was removed from 12 μg of total RNA of each sample using the Ribo-Zero Gold kit (Illumina, CA USA). The rRNA-depleted RNA samples were then polyadenylated using the SMARTer smRNA-Seq kit for Illumina (Clontech Laboratories, Inc., CA USA). The full-length cDNA was generated following the same protocols as those used for total RNA. After size selection with the ProNex beads (Promega, WI USA), the full-length cDNA libraries were constructed with the SMRTbell Express kit 2.0 (Pacific Biosciences, CA USA). The concentration and the size distribution of each library were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), respectively. Each library was then sequenced on the Sequel system (Pacific Biosciences, CA USA) using one SMRT Cell 1 M v3 LR with Binding Kit 3.0 and Sequencing Kit 3.0, with a collection time of 20 h.

RNA extracted from isolated human mature adipocytes were used to map the full-length transcript using Pacbio iso-seq method. The sample was prepared as described in “Procedure & Checklist – Iso-Seq™ Express Template Preparation for Sequel® and Sequel II Systems” (PN 101-763-800 Version 02 (October 2019)) https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Iso-Seq-Express-Template-Preparation-for-Sequel-and-Sequel-II-Systems.pdf. Briefly, using SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences) together with NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module and Iso-Seq Express Oligo Kit (Pacific Biosciences). 300 ng of total RNA was used for cDNA Synthesis followed by cDNA Amplification. The amplified cDNA then went into SMRTbell library construction. Finally, the sample was sequenced on Sequel II System using Sequel^® II Sequencing Plate 2.0 and On-Plate Loading Concentration of 80 pM. PacBio data were processed and evaluated with several tools in SMRT Analysis (v2.2.0 and v2.3.0) and in-house pipelines at theNational Genomics Infrastructure (NGI) /Uppsala Genome Center Sweden. The transcripts mapped to ADIPINT loci were extracted and presented in the current study. The complete sequences of the 5’ full length trascripts of ADIPINT is available upon request.

Full-length transcript sequencing followed the Isoseq Express Template Preparation protocol from Pacific Biosciences (Menlo Park, CA). Leaf or flower RNA samples were prepared by pooling an equal amount of four biological replicates of both control and MeJA-treated groups. Barcoded double-stranded cDNA libraries were generated using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module, the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, Ipswich, MA) and the Iso-Seq Express Oligo Kit (Pacific Biosciences). SMRTbell sequencing adapters were added to the cDNAs with the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences). The libraries were sequenced on a PacBio Sequel II sequencer (Pacific Biosciences) at UC Davis Genome Center.