title: GSM8740137: mouse intestine organoid, RNase5 knockdown, rep2; Mus musculus; ncRNA-Seq
library_construction_protocol: the crypt lysates were triturated by passing them through a 26-G needle ten times, and nuclei and debris were removed by centrifugation at 18,928 × g for 10 min. The supernatant was lysed using Trizol, and the RNA was isolated.Total RNA was diluted in Novex TBE-Urea sample buffer, heated to 65°C for 5 min and cooled briefly to room temperature prior to loading. A 15% polyacrylamide TBE-urea gel was prerun for 30 min, and samples were electrophoresed to the bottom of the gel at 100 V in 0.5× TBE running buffer. Following electrophoresis, the gel was equilibrated in 0.5× TBE for 5 min and stained with SYBR Gold solution diluted in 20 mL of 0.5× TBE buffer for 20 min with agitation, according to the manufacturer's instruction. Gels were imaged on a Gel Doc XR System (Bio-Rad) using UV illumination. Small RNAs with 10-50 nt fragment sizes were isolated by size excision on a 15% polyacrylamide TBE-urea gel. Before library preparation, the following treatments were carried out: 3′-aminoacyl (charged) deacylation to 3′-OH and 3′-cP (2′,3′-cyclic phosphate) removal to 3′-OH for 3′ adaptor ligation, 5′-OH (hydroxyl group) phosphorylation to 5′-P for 5′-adaptor ligation, and m1A and m3C demethylation for efficient reverse transcription. The small RNA library was prepared with the NEBNext Small RNA Library Prep Set for Illumina. The libraries were qualified and absolutely quantified using an Agilent BioAnalyzer 2100. For standard small RNA sequencing on an Illumina HiSeq 2500 instrument, the sequencing type was a 50-bp single read.
identifiers.namespace: GEO
identifiers.id: GSM8740137_r1