GSM1531868: dcl1dcl2_3_RNAseq; Colletotrichum higginsianum IMI 349063; RNA-Seq
Total RNA isolated from 4 day old mycelia using Trizol reagent. 10 µg of total RNA was treated with TURBO DNase I (Ambion). 1.5 µg of DNase-treated RNA was treated with RiboZero Magnetic Gold Kit-Yeast (Epicentre) to deplete ribosomal RNAs. Libraries were prepared according to Wang et al. (2011, PLoS One 6:e26426) and Carbonell et al. (2012, The Plant Cell Sep;24(9):3613-29). Briefly, 70 ng of RiboZero-treated RNA were fragmented at 94ºC for 7 min and reverse-transcribed using Superscript II (Invitrogen). Second strand synthesis was done using DNA-polymerase I (Enzymatics) in the presence of d-UTP for strand-specificity, and DNA was end-repaired using the End-Repair Mix (Enzymatics). The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo) (Enzymatics) and dATP to leave a protruding 3'A for ligation of Y-adaptors. DNA adaptor 1 (ACACTCTTTCCCTACACGACGCTCTTCCGATC*T) and DNA adaptor 2 (G*ATCGGAAGAGCGGTTCAGCAGGAATGCCGAG) were annealed to generate the Y-shape adaptors, and ligated to the 3' end. Before PCR amplification, dUTPs were degraded with Uracil DNA Glycosilase (Enzymatics). Twelve cycles of PCR amplification using the oligonucleotides PE-Primer-F (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and PE-Primer-R (series N701-N710) was performed to add index sequences the samples. DNA amplicons were analyzed with a Bioanalyzer (DNA HS Kit, Agilent) to ensure the PCR amplicon was ~250 nt then quantified using the Qubit HS Assay Kit (Invitrogen). PE-Primer-R Series N701-N710:  N701, CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTGACTGGAGTTCAGACGTGT; N702, CAAGCAGAAGACGGCATACGAGATCTAGTACGGTGACTGGAGTTCAGACGTGT; N703, CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTGACTGGAGTTCAGACGTGT; N704, CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTGACTGGAGTTCAGACGTGT; N705, CAAGCAGAAGACGGCATACGAGATGGACTCCTGTGACTGGAGTTCAGACGTGT; N706, CAAGCAGAAGACGGCATACGAGATTAGGCATGGTGACTGGAGTTCAGACGTGT; N707, CAAGCAGAAGACGGCATACGAGATCTCTCTACGTGACTGGAGTTCAGACGTGT; N708, CAAGCAGAAGACGGCATACGAGATCAGAGAGGGTGACTGGAGTTCAGACGTGT; N709, CAAGCAGAAGACGGCATACGAGATGCTACGCTGTGACTGGAGTTCAGACGTGT; N710, CAAGCAGAAGACGGCATACGAGATCGAGGCTGGTGACTGGAGTTCAGACGTGT
GEO Accession
GSM1531868