Life Technologies Proton

Proton Fastq Files (GEO: GSE48034)

Method

Starting Material, Enrichment and Library Construction

Libraries were prepared from 1 ug of MAQC A, B, C, and D RNA containing ERCC controls using either polyA enrichment (as described for PGM) or ribo-depleted using Ribo-Zero Magnetic Kit (MRZH11124, Epicentre). After enrichment, 8-9 ng of polyA mRNA or 150 ng of ribo-depleted RNA was used for ligation reactions. The size selection step was adjusted to 220 bp using a standard Pippin Prep protocol, generating library competent molecules with a template insert size of approximately 150 bp. The Ion Total RNA-seq Kit v2 (4476286 Rev D, Life Technologies) was used to prepare the MAQC libraries for sequencing.  The resulting material was quantified using the Agilent Bioanalyzer High Sensitivity Chip.

Ion Template Preparation and Sequencing

Emulsion PCR was performed using the One Touch 2 (OT2) system following the Ion P1 Template OT2 200 protocol (Life Tech 0007488 Rev2.0) by using 315-615 million DNA molecules post-library preparation.  Enriched spheres were quantified and approximately 400 to 800 million spheres were recovered.  P1 Chips were loaded according to the spinning protocol and sequencing was performed using the Proton 200 sequencing kit (MAN0007491 Rev 3.0).  Base calls were collected with Torrent Suite using v3.4.1 software.