title: cDNA-seq of C.cinerea: Young Fruiting Body Stipe (YS)
design: From each of the five purified total RNA samples, 2 g of RNA was used to prepare full-length cDNA libraries using the TeloPrime Full-Length cDNA Amplification Kit V2 (Lexogen, Vienna) protocol. Endpoint PCR was performed using the same kit with 12 to 18 cycles. The concentration and integrity of the samples were determined using a Qubit 2.0 fluorometer with the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific) and the Bioanalyzer 2100 (Agilent Technologies) with the DNA 7500 chip. The amplified cDNA samples were barcoded using the Oxford Nanopore Native Barcoding Expansion Kit (EXP-NBD104). The proportionally mixed barcoded samples were used to prepare libraries with the Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies), which were then sequenced on two MinION flow cells (FLO-MIN106 R9.4.1, Oxford Nanopore Technologies). Base calling and demultiplexing were performed using Guppy v3.2.4. Only reads with a Q value 7 were used in further steps. The base called reads were adapter trimmed and oriented using Pychopper v2.3.1