Pacific Biosciences (PacBio) RS

PacBio Fastq Files (Direct Download)

PacBio bas.h5 Files (Direct Download)


The RNA-seq methods used in this study for full length cDNA sequencing on the Pacific Biosciences RS are an early access protocol provided to the ABRF NGS consortium; refinements to the protocol are under development by the vendor.

Starting Material

MAQC A and MAQC B RNA samples were used to generate PacBio libraries.  The polyA+ fraction was purified from total RNA using Invitrogen Dynabeads Oligo(dT)25, according to the manufacturer’s protocol (61002, Life Technologies).  MAQC A RNA (100 µg) was mixed with 1.33 mg of washed Dynabeads, and 200 µg MAQC B RNA was mixed with 2.66 mg of washed Dynabeads, collected by magnetic precipitation, washed as directed, and eluted into 27 µl of 10 mM Tris-HCl.  The amount and purity of the polyA+ RNA was assessed using an Agilent 2100 Bioanalyzer RNA NanoChip. 

cDNA Synthesis

Full-length, double-stranded cDNA was synthesized from the polyA+ mRNA using the first five steps of the Invitrogen SuperScript Full Length cDNA Library Construction Kit II (A13268, Life Technologies); which included: (1) first strand cDNA synthesis; (2) RNase I treatment; (3) 5’G Cap-Antibody selection; (4) second strand cDNA synthesis; and (5) cDNA size fractionation by Sephacryl column chromatography and precipitation.  The cDNA was amplified with limited PCR using Phusion Hot Start Flex DNA Polymerase (M0535L, New England Biolabs), including PCR primers adapted from the 3’ oligo-dT primer used for first-strand cDNA synthesis and the 5’ adaptor used for second-strand cDNA synthesis, as described in the Invitrogen Superscript manual (primer sequences: pGGG ACA ACT TTG TCA AAG AAA and pTCG TCG GGG ACA ACT TTG TAC, respectively).  The PCR amplification profile was 98 °C for 2 min, 14 cycles x (98 °C for 0.5 min, 64 °C for 0.5 min, and 72 °C for 4 min) and a final extension at 72 °C for 4 min.  PCR-amplified cDNA was purified using 0.6x volume of Agencourt AMPure PB Beads (Beckman Coulter, Life Sciences Division), as specified by the supplier (Pacific Biosciences).  The purified cDNA was recovered in 50 µl of TE buffer.

Total cDNA was divided into three MW size classes of 1-2 kb, 2-3 kb, and 3-8 kb using SYBR green and 0.8% agarose gel size fractionation, and recovered using Qiagen QIAquick Gel Extraction (Qiagen).  Each cDNA size class was re-amplified using PCR conditions identical to those listed above.  In order to prepare enough cDNA for multiple sequencing library constructions, a total of eight 100 µl PCR reactions were performed in parallel for each of the three cDNA size classes.  The resulting PCR product was purified using 0.6x volume of AMPure PB beads as described above.  Purified cDNA was quantified by a fluorometric Qubit assay and evaluated using an Agilent 2100 Bioanalyzer DNA 12000 chip.

Library Construction and Sequencing

Each of the three size-fractionated cDNA pools from the MAQC A and MAQC B samples were distributed to three core laboratory sites for library preparation and data generation, resulting in a total of 18 libraries.  SMRTbell libraries were prepared from 0.5 and 1.0 µg of each cDNA size class according to the PacBio Large Insert Template Library Prep Kit.  Double stranded cDNA was subjected to DNA damage repair, end repair, and blunt-end ligation to hairpin adaptors.  Incomplete SMRTbell templates were degraded with a combination of Exonuclease III and Exonuclease VII.  Intact cDNA SMRTbells were purified by three sequential AMPure PB purifications.  Sequencing primers were annealed to the SMRTbell templates and subsequently bound to the sequencing polymerase using the Pacific Biosciences DNA/Polymerase v 2.0 binding kit, following manufacturer’s instructions.  The samples were sequenced on the PacBio RS using “C2” chemistry with SMRTcell loading via diffusion.  The data collection times were adjusted per cDNA size bin.  For size bins less than 2 kb, the 2 x 45 minute movie protocol was used, while bins at or above 2 kb used a 1 x 90 minute movie protocol.