5 protocols
Illumina pipeline export files were transformed into fastq format by applying this Unix command: awk '{print @$1_$2:$3:$4:$5:$6#$7/$8\n$9\n+\n$10}' file_export.txt > file.fastq
nucleic acid sequencing protocol
Aliquots of up to 5000 RT-PCRs reactions were pooled together and the sequencing library was prepared with no fragmentation using the TruSeq DNA protocol. DNA fragments were sequenced on a Illumina HiSeq2000 sequencer and 2 multiplexed libraries were loaded per lane. The sequencing was performed at the University of Lausanne, Lausanne, Switzerland.
nucleic acid extraction protocol
Human polyA+ RNAs were purchased from Clontech. The RNAs were isolated from tissue samples using a modified guanidium thiocynate extraction method followed by polyA+ RNA selection with two rounds of oligo(dT)-cellulose columns.
nucleic acid library construction protocol
First-strand cDNA samples were prepared from human poly(A)+ RNAs (BD-Clontech) with the SuperScript III kit (Invitrogen). Amplifications were performed in a final volume of 12.5 L with JumpStart REDTaq ReadyMix (Sigma-Aldrich) and a primer concentration of 0.4 M in 384-well plates format on an automatized Evoware platform (TECAN) combined with a Tetrad2 thermocycler (Bio-Rad) that allows processing four plates in parallel. Because monoexonic amplification is sensitive to genomic DNA contaminations, monoexonic models were assessed by amplification of cDNA in which a dNTP analog was incorporated using the mRNA Selective PCR Kit (TAKARA).
We experimentally assessed three categories of gene models: (1) spliced models in which one primer could be placed within 75 nt of a junction and that will result in about half of the sequencing reads covering the junction (“Multi-span”); (2) spliced models in which this was unfeasible (“Multi”); and (3) monoexonic genes (“Mono”). In the case of “Multi-span” primers, the “junction primer” is positioned within an exon not more than 65 bp away from the targeted junction to ensure that sequencing reads will cross the junctions with a minimum of 10 nt, while the second primer maps within an adjacent exon. To increase the fraction of junctions we could possibly test, we then designed primer pairs further away from the junction (“Multi” primers). Primers were designed using Primer3. We used parameters minimizing the formation of primer dimers and maximizing primers “stickiness”. Primer pairs were further filtered for mapping within repeat-regions and alternative priming within 30 kb in duplications and paralogous sequences (maximum of two tolerated mismatches).