title: GSM8791788: Mettl14KO_NPC,replicate2,RNA-seq; Mus musculus; RNA-Seq
library_name: GSM8791788
library_construction_protocol: Total RNA fraction was isolated from cultured hypothalamic NPCs samples using RNA Clean & Concentration Kit. Enrichment of polyadenylated RNA from total RNA was performed using Oligo(dT) dynabeads according to the manufacturer's protocol. The polyadenylated RNA was chemically fragmented into ∼80-nt-long fragments using zinc fragmentation reagent. To purify the RNA, 20 μl MyOne Silane Dynabeads were washed in 100 μl RLT, resuspended in 30 μl RLT, and added to the eluted RNA. 60 μl 100% ethanol was added to the mixture, the mixture attached to the magnet and the supernantant discarded. Following two washes in 100 μl of 70% ethanol, the RNA was eluted from the beads in 16.5 µl water. 50 ng of polyadenylated RNA fragments from Mettl14 knockout or control NPCs were used for cDNA library preparation with the NEBNext Ultra II RNA Library Prep Kit (NEB, E7770S) following the manufacturer's protocol.Then the IP samples were used for cDNA library preparation with the NEBNext Ultra II RNA Library Prep Kit. Sequencing library fragment sizes were quantified by bioanalyzer with an average size of ∼400 bp, and concentrations were quantified by qPCR (KAPA). Samples were pooled, loaded at 2.7 pM, and sequenced on a Next-Seq 500 (Illumina).