title: GSM7699286: CytoRP_60m_2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Frozen cells on nitrocellulose membranes were placed in metal casings with frozen lysis buffer to a total of 4 g. Samples were harvested four at a time by placing two casings containing separate samples in a mixer mill and grinding for 3 minutes at 15 Hz, while two other casings were submerged in liquid nitrogen. Casings were swapped for six rounds of grinding, after which samples were scraped into 50 mL eppendorf tubes with a pre-chilled spatula. Lysates were pre-cleared by spinning at 3000xg for 5 minutes at 4oC, supernatant was transferred to a fresh eppendorf tube and then cleared by spinning at 20,000xg for 10 minutes at 4oC. Samples were distributed into 250 μL aliquots and flash frozen in liquid nitrogen Gel-purified RNA was treated with PNK and ligated to a pre-adenylated adapter (5′ /5rApp/NNNNNNCTGTAGGCACCATCAAT/3ddC/3′). Superscript III was used for reverse transcription (with primer 5′ /5Phos/GATCGTCGGACTGTAGAACTCTGAACCTGTC/iSp18/CACTCA/ iSp18/CAAGCAGAAGACGGCATACGAGATATTGATGGTGCCTACAG 3′) and circ ligase for circularization. Illumina adapters were introduced by 10-14 cycles of PCR.