title: GSM8577453: HUVEC small RNA, VEGFA165 IP, rep3; Homo sapiens; ncRNA-Seq
library_construction_protocol: Cell pellets were directly lysed in 500 µL of RNAzol RT (Molecular Research Center). Samples were shaken at 50°C for 5 minutes. To phase separate the RNA, 0.4X volumes of water was added, vortexed, let to stand for 5 minutes at 25°C and lastly spun at 12,000x g at 4°C for 15 minutes. The aqueous phase was transferred to clean tubes and 1.1X volumes of isopropanol was added. The RNA was then purified over a Zymo column (Zymo Research). For all column cleanups, we followed the following protocol. First, 350 µL of pure water was added to each column and spun at 10,000x g for 30 seconds, and the flowthrough was discarded. Next, precipitated RNA from the RNAzol RT extraction (or binding buffer precipitated RNA, below) was added to the columns, spun at 10,000x g for 20 seconds, and the flowthrough was discarded. This step was repeated until all the precipitated RNA was passed over the column once. Next, the column was washed three times total: once using 400 µL of RNA Prep Buffer (3M GuHCl in 80% EtOH), twice with 400 µL of 80% ethanol. The first two spins were at 10,000x g for 20 seconds, the last for 30 seconds. Libraries made following MSR-seq, PMID: 35513407