Step 1: Precipitation of total RNA (residual salt removal)
- Add 0.1 volume of 5 M Ammonium Acetate to 5-50 ug of testis RNA.
- Add 1 μL Glycogen.
- Add 2.5 volumes of 100% ethanol.
- Mix thoroughly by vortexing.
- Precipitate at –20°C overnight.
- Recover the RNA by centrifugation at 12,000 x g for 30 min at 4° C.
- Carefully remove and discard the supernatant.
- Centrifuge the tube briefly for the second time and aspirate away any additional fluid.
- Add 1 mL 70% ethanol, and vortex the tube a few times. Repellet the RNA by microcentrifuging, for 10 min at 4°C. Remove supernatant carefully not to disturb the RNA pellet.
Step 2: Isolation of poly(A) RNA using Poly(A) Purist MAG (Catalog Number: AM1922, Publication Number: 1922, Revision D)
Step 3: Generation of first-strand cDNA using Clontech SMARTer PCR cDNA synthesis kit (Catalog Number:634926, Protocol Number: PT4097-1, Version number: 040114)
Step 4: Generation of double-strand cDNA using Clontech SMARTer PCR cDNA synthesis kit (Catalog Number: 634926, Protocol Number: PT4097-1, Version number: 040114)
- PCR Cycle Optimization for Large-Scale PCR using KAPA HiFi Enzyme
Generation of large-scale double-stranded DNA using the optimized cycle number.
Step 5: RT-PCR primer design for capturing nine ampliconic gene transcript families from human males
For each of the samples, a unique PacBio barcode is added to the forward and the reverse primers according to Guidelines for Using PacBio Barcodes for SMRT Sequencing (http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf). This is important for the differentiation of transcripts coming from different samples.
Step 6: Gradient PCR on human testis cDNA to determine the optimal annealing temperature for each RT-PCR primer pair specific to each multigene transcript family
Nine ampliconic gene transcript family targets were amplified using a range of annealing temperatures via Gradient PCR (55-68°C). Subsequently, RT-PCR products were separated with electrophoresis on a 0.8% agarose gel and visualized under the UV light. Distinct bands on the gel allowed us to determine the optimal annealing temperature for capturing each of the multigene transcript family targets (See Table 2).
Step 7: Final PCR amplifications of each multigene transcript family targets using a specific annealing temperature (See Table 2), PCR protocol (See Table 3) and RT-PCR product purification using AMPure XP beads
Step 8: Preparation of RT-PCR products for PacBio sequencing (RSII)
Multigene family RT-PCR products were processed in two groups. Equimolar amounts of RT-PCR products specific to each multigene family and of size less than 1 kb are pooled to the total amount of 2 ug, from which one PacBio SMRTbell library is prepared and sequenced using one SMRT cell using PacBio’s Procedure & Checklist – Isoform Sequencing (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection (https://www.pacb.com/wp-content/uploads/2015/09/Procedure-Checklist-Isoform-Sequencing-Iso-Seq-using-the-Clontech-SMARTer-PCR-cDNA-Synthesis-Kit-and-No-Size-Selection.pdf). Equimolar amounts of RT-PCR products specific to each gene family and of size greater than 1 kb and smaller than 2 kb have been pooled to the total amount of 2 ug, from which one PacBio SMRTbell library has been prepared and sequenced using one SMRT cell.
Step 9: Preparation of RT-PCR products for Illumina sequencing
Nextera XT libraries were constructed from the same RT-PCR products using Nextera XT DNA Sample Preparation Guide (Part# 15031942, Revision C). Equal volumes of bead-normalized libraries are pooled and sequenced on a MiSeq instrument using one MiSeq Reagent Nano Kit, v2 (250x250 paired-end sequencing).