title: Gm12878-pacBio
design: cDNA Synthesis  Poly-A-Selected RNA from the same batch as for the short-read library was used to synthesize unfragmented cDNA using Life Technologies’ Superscript double-stranded cDNA synthesis kit (catalogue #11917-010) and reverse transcription (RT) was primed with an anchored oligo(dT)20 primer (Life Technologies catalogue #12577-011). The cDNA was purified using Qiagen MinElute PCR purification kit (catalogue #28004) and eluted with 50µL of RNase-free water. The cDNA was quantified using Qubit HS dsDNA kit (Life Technologies catalogue #Q32851) and quality was assessed using the Agilent 2100 bioanalyzer.     Library-preparation, Sequencing and Data Collection  SMRT bell libraries were generated using Pacific biosciences’ 1.0 template prep kit (part #001-322-716) and 2.0 template prep kit (part# 001-540-726) and Pacific Biosciences’ template preparation and sequencing protocol for 0.25-3kb libraries. SMRT bell templates were bound to polymerases using the DNA/polymerase binding kit XL 1.0 (part #100-150-800) and v2 primers. Polymerase-template complexes were bound to magbeads using Pacific Biosciences’ Magbead binding kit (part #100-134-800) and sequencing was carried out on the Pacific Biosciences’ real-time sequencers RT and RTII using C2 sequencing reagents. Movie lengths were 55 minutes and two movies were observed for each SMRT cell on the RT and 120 minutes with one movie on the RTII. Sub-read filtering was performed using Pacific Biosciences’ SMRT analysis software (v1.3.3).