Ga flow cell

mRNA was isolated from total RNA using Oligo-dT beads (Invitrogen) and then fragmented by heating at 94 °C. First-strand complementary DNA (cDNA) was synthesized with random hexamer primers and second-strand cDNA synthesized with DNA polymerase. Double-stranded cDNA was end-repaired/blunt-ended with Klenow polymerase, T4 DNA polymerase, and T4 polynucleotide kinase. A single adenosine moiety was added to the cDNA using Klenow exo^- and dATP. All the enzymes used above were included in the kit (mRNA-Seq Sample Pre Kit) from Illumina. Illumina adapters (containing primer sites for sequencing and flowcell surface annealing) were ligated onto the blunt-ended cDNA, and the library of DNA fragments (200–250 bp) was separated from unligated adapters by gel excision. Libraries were amplified by PCR with Phusion polymerase, denatured with 100 mM sodium hydroxide, and diluted in hybridization buffer before they were loaded onto a single lane of an Illumina GA flowcell. Cluster formation, primer hybridization, and paired-end sequencing (101 × 2 cycles) were performed using Illumina reagents. Expression level was determined using the mapped reads per kilobase of exon per million mapped reads (RPKM) in the spore cells.

The genome of the lichen-forming fungus E. pusillum was sequenced using high-throughput next-generation sequencing technology and the sequencing platforms were Roche 454 and Illumina Solexa systems. Genomic libraries containing 8-kb inserts were constructed and 1,394,086 paired-end reads (281.9 Mb) were generated using the 454 Roche GS FLX system. The Illumina adaptors were ligated onto the genomic DNA fragments, and DNA fragments with estimated sizes of 0.5 kb to 3 kb were selected using gel-electrophoresis. Libraries were PCR-amplified using Phusion polymerase. Sequencing libraries were denatured with sodium hydroxide and diluted in hybridization buffer for loading onto a single lane of an Illumina GA flow cell. A Solexa sequencer generated the mate-paired reads (7,155,072 reads, 716 Mb) and paired-end reads (18,176,986 reads, 1818 Mb). Solexa sequencing paired-end reads and mate-paired reads were assembled by SOAPdenovo [97 (link)], which adopts the de Bruijn graph data structure to construct contigs.

The single-stranded cDNA libraries were constructed as described in [44 (link)]. Gel electrophoresis was used to select for DNA constructs approximately 200 bp in size, and amplified by PCR. The libraries were loaded onto two separate lanes of an Illumina GA flow cell and sequencing reactions performed according to the manufacturer’s recommended protocol.