Transcriptor First Strand cDNA Synthesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 0.5 ul (10 U) of Reverse Transcriptase to the reaction mix and incubate for Incubate 30 min at 55°C. Inactivate Transcriptor Reverse Transcriptase by heating to 85°C for 5min

Protocol tips
Add 0.5 ul (10 U) of Reverse Transcriptase to the reaction mix and incubate for Incubate 30 min at 55°C. Inactivate Transcriptor Reverse Transcriptase by heating to 85°C for 5min

Publication protocol

Yeast cells (107) in mid-log phase were collected for total RNA extraction using the MasterPure yeast RNA purification kit (Epicentre). RNA was then digested with TURBO DNA-free (Life Technologies) to eliminate contaminating DNA and purified using RNeasy Kit (Qiagen). RNA concentration was measured using NanoDrop 2000 UV–vis Spectrophotometer (Thermo Scientific). To obtain sense or antisense strand-specific complementary DNA, total RNA (1–2 μg) was RT using the Transcriptor first-strand complementary DNA synthesis kit (Roche) and a reverse strand primer listed in Supplementary Table 2. To measure SER3 and CHA1 transcript levels, total RNA was reverse transcribed using oligo-dT primer. Transcript levels were assessed using qPCR in a Bio-Rad CFX Connect Real-Time PCR Detection System employing both forward and reverse primers (Supplementary Table 2). Fold-change in sense or antisense transcript level in a mutant was calculated using the 2−ΔΔCT method relative to the respective transcript level in the WT control. Four independent biological replicates and triplicate PCR were performed.

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