Multiplexing sample preparation oligo kit

RNA was extracted using the Qiagen RNeasy Plant Mini Kit (Qiagen), with an additional sonication step after addition of buffer RLT. RNA integrity was checked using the Agilent BioAnalyzer 2100 (Agilent). Approximately 2 µg of total RNA was used for mRNA-Seq library construction according to the manufacturer’s recommendations (Illumina).
We used the multiplexing sequencing adapters provided in the Multiplexing Sample Preparation Oligo Kit (Illumina). Size selection of the library was performed on a 2% agarose gel (Low Range Ultra Agarose, Biorad 161-3107). The denatured library was diluted to a final concentration of 6 pM and loaded on a paired-end read flow cell (TruSeq v5 kit, Illumina). To minimize lane effects the samples were multiplexed. Each sample was sequenced in duplicate in 2 different lanes (4 lanes total with 8 MID tags per lane). After cluster generation, the multiplexed library was sequenced on an Illumina Genome Analyzer IIx (36 cycles, paired end).

Multiplexed paired-end libraries were constructed from 5 μg of genomic DNA purified using the Purgene kit (Qiagen). The genomic DNA was sheared by sonication and end-repaired using the End-it DNA End-repair kit (Epicentre Technologies). Common adaptors from the Multiplexing Sample Preparation Oligo Kit (Illumina) were then ligated to the genomic DNA fragments, and the fragments were then subjected to 18 cycles of amplification using the Library Amplification Readymix (KAPA Biosystems). The amplified products were fractionated on an agarose gel to select 600 bp fragments, which were subsequently sequenced on an Illumina HiSeq 2000 using the Illumina GAII sequencing procedure for paired-end short read sequencing. Reads from each read pair were mapped separately by bowtie version 2.2.1 [108 (link)] to a reference sequence that contained revision 64 of the S. cerevisiae S288c genome [109 (link)], hisG from Samonella enterica, and the kanMX4 marker (S3 Table). Reads are available from National Center for Biotechnology Information Sequence Read Archive under accession number: SRP107803.

Multiplexed paired-end libraries were constructed from 5 µg of genomic DNA purified using the Purgene kit (Qiagen). The genomic DNA was sheared by sonication and end-repaired using the End-it DNA End-repair kit (Epicentre Technologies). Common adaptors from the Multiplexing Sample Preparation Oligo Kit (Illumina) were then ligated to the genomic DNA fragments, and the fragments were then subjected to 18 cycles of amplification using the Library Amplification Readymix (KAPA Biosystems). The amplified products were fractionated on an agarose gel to select 600 bp fragments, which were subsequently sequenced on an Illumina HiSeq 2000 using the Illumina GAII sequencing procedure for paired-end short read sequencing. Reads from each read pair were mapped separately by bowtie version 0.12.7 [74] (link) to a reference sequence that contained revision 64 of the S. cerevisiae S288c genome (http://www.yeastgenome.org), hisG from Samonella enterica, and the hphMX4 marker (Table S1). Sequencing data have been deposited at NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under the accession SRP039033.