GSM5090157: Spt4 NETseq Replicate 3; Saccharomyces cerevisiae; OTHER
Extraction methods were based on Clarke et. al., NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I, PNAS, 2018. 115 (50), E11633-E11641. In brief, cells were harvested by filtration and flash frozen. Then, cells were lysed for 10-one minute cycles with a mikro dismembranator II under cryogenic conditions. Immnoprecipitation was performed as previously described, by incubating samples with anti-HA magnetic beads for 3 hours. RNA was extracted with an acidic phenol-chloroform extraction, then RNA was precipitated at -80ºC with ammonium acetate ethanol solution. Libraries were constructed based on previous methods in  Clarke et. al., NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I, PNAS, 2018. 115 (50), E11633-E11641. To summarize, a DNA linker containing a UMI sequence (5'-/5rApp/CANNNNNNNNCTCCACGAGTCATCCGC /3ddC/-3') was ligated to the 3' end of the isolated RNA transcripts. A gel extraction was performed to remove residual linker from the sample. Samples underwent reverse transcription to produce cDNA, then another gel extraction was used to remove excess reverse transcription primer from sample. Finally, samples were circularized, libraries were amplified, and PCRCleanDX magnetic beads were used to prepare libraries for sequencing.