GSM6127818: Cgset4Δ grown in CAA medium: Replicate-2; [Candida] glabrata; RNA-Seq
Total RNA was extracted using the acid-phenol method. Briefly, caspofungin-treated and untreated cultures of C. glabrata wild-type and Cgset4Δ stains were pelleted down and washed twice with chilled DEPC-treated water. The cell pellet was suspended in 350 μl of chilled AE solution (50 mM NaOAc and 10 mM EDTA) and lysed by addition of 40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) and vortexing for 10 sec. Next, the samples were incubated at 65oC for 15 min on a thermomixer followed by incubation on ice for 5 min. The samples were centrifuged at 4oC at 13,000 rpm for 10 min, and the upper aqueous layer was collected in a new 1.5 ml microcentrifuge tube. 400 μl of chloroform was added to the aqueous layer and centrifuged at 3,000 rpm for 10 min at 4⁰C. The aqueous layer was transferred to a 1.5 ml microcentrifuge tube, and RNA was precipitated with 40 µl of 3 M sodium acetate (pH 5.3) and 400 µl of ethanol, following incubation at -20oC for 20 min. Next, RNA was pelleted down by centrifugation at 13,000 rpm for 10 min at 4oC, and the pellet was washed with chilled 70% ethanol, air dried and suspended in 100 μl DEPC-treated water. To remove DNA contamination, 1 μl of DNase I was added to 1 µg of total RNA and incubated for 20 min at 37°C. RNA-Seq library was prepared with 1 μg of total RNA using TruSeq RNA Sample Prep Kits (Illumina), as recommended by the manufacturer. In brief, total RNA was subjected to poly-A tail-based mRNA purification using magnetic beads attached with poly-T oligos. After purification, mRNA was fragmented using divalent cations at high temperature. The cleaved RNA fragments were used to synthesize cDNA first strand using reverse transcriptase and random primers. The synthesis of the second strand cDNA was done using DNA polymerase I and RNase H. These cDNA fragments underwent to end repair process followed by addition of a single 'A' base and ligation of the adaptors. The product was finally purified and enriched with PCR to create final cDNA library. Bioanalyzer plots were used to assess mRNA quality, enrichment success, fragment size, and the final library size. Both qubit and qPCR methods were used to measure the quantity of library before sequencing.