anchor: NlaIII tag length: 22 tag count: 295439 cultivar: Kakehashi age: 30-day-old tissue: leaf
Extracted molecule
total RNA
Extraction protocol
ds-cDNA was synthesized using biotinylated dT primer. After digestion of cDNA with anchoring enzymes (NlaIII, DpnII or BfaI), digested fragments were associated with streptavidin magnetic beads. Adapters were ligated to cDNA ends on the beads and adapter-cDNA was digested with EcoP15I. Released fragments after enzyme digestion were ligated to adapter encoding barcode. Obtained fragments were once amplified PCR and applied to sequence analysis.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
cDNA
Instrument model
Illumina Genome Analyzer II
Description
Adapter-tags were amplified by ten cycles of PCR reaction.
Data processing
Sequece reads were sorted by barcode into separate files. From each read in each file, 26-bp sequence upstream anchoring enzyme site (CATG, GTAC or CTAG) was extracted. Extracted 26-bp sequences (tags) were count. These process was done by script written in Perl. Tags were extracted from sequence data of multiplexed samples, which were analyzed by Illumina Genome Analyzer IIx.
