Qiaquick pcr kit

Total RNA was extracted using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent, CA, USA). Subsequently, rRNA was eliminated, but mRNA and non-coding RNA (ncRNA) were kept. With the use of fragmentation buffer and random primers, the enriched mRNA and ncRNA were broken up into small pieces and then reverse transcribed into cDNA. Using DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP), and buffer, a second-strand cDNA was created. The cDNA fragments were then purified and end-repaired. They were poly (A) tailed and ligated to Illumina sequencing adapters using the QiaQuick PCR kit (Invitrogen, CA, USA). The UNG (uracil-n-glycosylase) enzyme was used to digest the second-strand cDNA. The digested products were analyzed using agarose gel electrophoresis, and then sequenced using Illumina HiSeqTM4000 sequencing by Gene Denovo Biotechnology (Guangzhou, China).

A PCR fragment was amplified from FGSC 2489 genomic DNA with the primers NCU00378_XbaI_F and NCU00378_PacI_R (Additional file 1: Table S8) using Phusion DNA polymerase (NEB # M0530). The final product was subjected to PCR purification with Qiaquick PCR kit (Catalog # 28104) and cloned into the pCR^® vector (Invitrogen # 44-0302) according to the manufacturer’s protocol. The NCU00378 insert was excised from the vector with XbaI and PacI and cloned into a modified pMF272 vector cut with XbaI/PacI in frame with an eGFP reporter ORF and flanked by tef-I promoter and ccg-1 terminator sequences from pMF272 [72 (link)]. The deletion strains for NCU00378 (FGSC 12919) were crossed with FGSC 6103 to create an NCU00378 deletion in a his3⁻ background. Progeny were genotyped by Phire Plant Direct PCR (Thermo Scientific # F-130WH) on conidia with the primers Hphp and NCU00378_3r (Additional file 1: Table S8).
Transformation was performed by electroporation according to a protocol available at the FGSC or as modified by Ziv and Yarden [73 (link)]. Conidia from the selected transformants were genotyped by PCR for the hygromycin cassette and also for the NCU00378-GFP construct with the primers NCU00378_F and pMF272_Rev (Additional file 1: Table S8).