GSM8759369: Rrm4mR3-Gfp RNAseq replicate3; Mycosarcoma maydis; RNA-Seq
To purify total RNA, 50 ml of hyphal cell culture with an OD600 of 0.5 was used. The cells from the 50 ml culture were collected and resuspended in 2 ml of 1x PBS, divided, and transferred into two 2 ml reaction tubes (1 ml each). The tubes were centrifuged at 16,200 g for 5 minutes at 4°C, and the supernatant was completely removed. The cell pellets were flash-frozen in liquid nitrogen. A 5 mm steel bead was added to each 2 ml tube, which were then placed in the pre-cooled TissueLyser Adapter (Qiagen). The samples underwent two rounds of mechanical disruption for 30 seconds at 30 Hz each, with a cooling step in liquid nitrogen between rounds.The cryogenically frozen cell powder was resuspended in 500 µl of TriZol (Invitrogen, 15596026) and incubated in a thermo block at room temperature for 5 minutes at 1000 rpm. Next, 500 µl of RNase-free water was added, and the mixture was manually shaken for 30 seconds. Subsequently, 400 μl of chloroform/isoamyl alcohol (24:1- BioChemica A1935,0500) was added. The samples were shaken vigorously by hand for 3 minutes, then incubated at room temperature for another 3 minutes. Following this, the samples were centrifuged for 10 minutes at 16,200 g. The aqueous phase was transferred to a fresh 1.5 ml tube pre-filled with 250 μl of chloroform/isoamyl alcohol (24:1- BioChemica A1935,0500). This extraction process was repeated, followed by centrifugation at 16,200 g for 5 minutes at 4°C.The aqueous phase was transferred to a solution containing 40 μl of sodium acetate (3 M, pH 5.2) and 1 ml of ice-cold ethanol (100%), then precipitated at -20°C for 2 hours. The RNA pellet was collected by centrifugation for 30 minutes at 16,200 g and 4°C, washed twice with 75% ice-cold ethanol, and centrifuged for 5 minutes at 16,200 g and 4°C. The pellet was briefly dried and resuspended in 50 μl of RNase-free water. RNA concentration was measured and adjusted accordingly. Ribosomal RNA was depleted,   and the residual RNA was reverse transcribed into cDNA using the TruSeq library preparation kit with Ribo-Zero Gold (Illumina). Sequencing was performed on an Illumina NextSeq 500, generating 150-nt single-end reads.