title: GSM8681911: LN229 cells, LN2; Homo sapiens; miRNA-Seq
library_construction_protocol: Small RNAs were extracted from Ac4ManNAz-labeled U87, LN229, and Hela cells by using RNAzol RT kit (Molecular Research Center, RN-190). The extracted small RNAs were treated with DBCO-biotin and then purified through Zymo columns. The MyOne C1 Streptavidin magnetic beads (Thermo Fisher Scientific, REF: 65001) were utilized to enrich Ac4ManNAzlabeled glycoRNAs. MyOne C1 streptomycin avidin magnetic beads containing 50 ng/mL glycogen were incubated in 500 μL Biotin Wash Buffer at 25 ℃ for 1h, then centrifuged at 3000 rpm at 4℃ for 2 min, and the supernatant was discarded. The magnetic beads were re-suspended in 500 μL Biotin Wash Buffer, added into small RNAs, rotated and mixed at 4 ℃ for 2 h, and centrifuged at 3000 rpm for 2 min. The supernatant was collected, and purified using Zymo columns. The magnetic beads and the small RNAs bound to them were re-suspended in solution containing 0.75M NaCl, 10 mM EDTA, and 10 mM Tris-Cl (pH 7.4) and incubated at 22 °C for 5 min. The above mixture was mixed with 1× volume of 5% B-mercaptoethanol (BME) solution, incubated at 22 °C for 5 min, and centrifuged at 3000 rpm for 2 min. The glycoRNA-rich supernatant was collected and purified by Zymo columns. RNA quality was checked using Agilent 4200 TapeStation (Agilenttechnologies, Santa Clara, CA, US) according to standard procedures. Sequencing libraries were constructed by QIAseq miRNA Library Kit for Illumina (Cat #331505, Qiagen) according to manufacturer's instructions. The sequencing was performed on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA).