Illumina HiSeq 2000/2500 and MiSeq

Illumina Fastq Files (GEO: GSE48035)

Methods

Starting Material and Enrichment

TruSeq libraries were synthesized from 2 ug of intact (RIN >7) and degraded (RIN <3) MAQC A and MAQC B RNA.  Three replicate libraries of intact A and B were prepared at each of three core laboratory sites.  Degraded RNA libraries were prepared at two additional core laboratory sites as listed in Table 2 (main text).  Ribosomal RNA-depletion material was generated using the Ribo-Zero Gold system (Epicentre Biotechnologies) according to the manufacturer’s instructions.  The preparation of three types of artificially degraded MAQC A and B RNA was performed at a single core laboratory site, using 75 ug of each RNA at a concentration of 1 ug/ul.  Three techniques were used: (1) heat treatment in deionized water at 99 oC for 10 min; (2) exposure to 1 ng/ul RNase A for a sufficient time period to result in a RIN of 3, with the RNase then neutralized with 10 ul RNase Inhibitor (RiboLock EO0381, Thermo Scientific); and (3) sonication within a Covaris S2 MicroVial for 6 x 55 sec at 5% DC, intensity 3, and 100 c/b.  All resulting RNA sample degradations (i.e., RIN values) were analyzed using the Agilent 2100 Bioanalyzer (Supp. Methods Fig. 2).

Library Construction and Sequencing

Following ribo-depletion, all recovered RNA was processed using the Illumina TruSeq RNA Sample Preparation Kit v2 protocol at the “elute-fragment-prime” step, followed by the standard TruSeq protocol.  Completed libraries were evaluated by DNA quantitation and Bioanalyzer analysis, and then submitted to a single core laboratory site for sequencing.  Sequencing libraries were constructed with barcodes to allow multiplexing of 12 samples per lane, pooled to target 200 million clusters per channel and 100 million reads per library, and distributed over multiple channels of three flow cells to normalize for lane and run variability.  Sequencing was carried out on Illumina HiSeq 2000 and 2500 instruments using protocols HCS 1.5.15.1 and RTA 1.13.48 and paired-end 50 bp reads.  One of the replicate libraries for intact MAQC B was also sequenced on a MiSeq instrument using the 4nM protocol (v2.2.0.2) targeting 15 million clusters with paired-end 250 bp reads.  The recently released TruSeq RNA Sample Preparation Kit v3 differs from v2 by including ribosomal RNA depletion and preserves cDNA strand orientation.  Lab site C compared polyA enriched and ribo-depleted libraries constructed using the v3 kit.