alias: GSM1673641
title: GSM1673641: wild type NET-seq; Saccharomyces cerevisiae BY4741; OTHER
library_construction_protocol: For NET-seq frozen cells were cryogenically pulverized for lysis and nascent RNA was isolated via an immunoprecipitation at 4°C of Rpb3-3xFLAG and purified using the miRNeasy kit (Qiagen, 217004). For RNA-seq total RNA was extracted using standard hot phenol extraction from 2.34x10E8 cells and resuspeded in 30uL of RNase free water. 3uL A 1:10 dilution of ERCC RNA standards from Life Technologies was added to 5uL of total RNA. For NET-seq a preadenylated RNA linker was ligated onto the 3' end of the immunoprecipitated RNA. Fragmentation of the ligated samples by alkaline hydrolysis followed by a size selection after electrophoresis on a polyacrylamide gel allowed for the final DNA library to contain inserts of a narrow range. cDNA synthesis by reverse transcription (SuperScriptsIII (Invitrogen)) was performed with a long 5' phosohorylated primer split by two 18 carbon spacers. Circularization of the RT product was performed with CircLigase (Epicentre) according to the manufacturer's directions. The PCR was performed directly on the circularized product resulting in DNA with Illumina cluster generation sequences on each end and a sequencing primer binding site positioned so that sequencing would start at the 3' end. DNA was purified from a PCR reaction that had not reached saturation (~8-10 cycles) by band extraction from a polyacrylamide gel. DNA was sequenced on the Illumina NextSeq 500 according to the manufacturer's instructions in 75 base pair reads. For RNA-seq total RNA after rRNA depletion was fragmented by alkaline hydrolysis followed by size selection after electrophoresis on a polyacrylamide gel.RNA was treated with polynucleotide kinase (NEB) followed by ligation to a preadenylated RNA linker. The remainder of the library construction was done as described inte NET-seq library construction. All samples were sequenced using an Illumina NextSeq 500 in 75 base pair reads
identifiers.namespace: GEO
identifiers.id: GSM1673641