GSM3389532: Untreated control SOD1 yeast library grown in YPD Biological Replicate B; Saccharomyces cerevisiae; RNA-Seq
Hot acidic phenol extraction of total RNA, followed by purification on Qiagen Rneasy columns with Dnase I treatment Hybridization capture and library construction: 500 ng of pooled barcoded DREADS libraries were blocked with 5 mg Cot-1 DNA (Invitrogen) and 1 ml each of p5 and p7 custom blocker small RNA (IDT).  Hybridization of a locked nucleic acid probe (5'-/5Biosg/AGTAAGCGCAT+CAATT+CAT+TC-3') specific to SOD1 (Exiqon) was carried out using the xGen hybridization and wash kit (IDT) according to the manufacturer's instructions, except that the hybridization and was carried out at 55˚C for 18 hours.  Subsequent capture, washes and purification were performed exactly as described in the kit's user manual, with the exception of all the steps that require incubation at 65˚C were done at 55˚C.  Captured fragments were eluted from the beads in 50 ml EB for 5 minutes 75˚C and amplified by 15 cycles of PCR with Illumina p5 and p7 primers.  Libraries were purified by 8% PAGE and subjected to deep sequencing using the DREADS/SHREADS library protocol as described above.