title: GSM8791802: Control_NPC,replicate1,m6A-seq; Mus musculus; OTHER
library_name: GSM8791802
library_construction_protocol: Mouse NPCs were isolated from E14.5 Mettl14f/f mouse embryonic hypothalamus using papain solution and cultured in DMEM/F12 containing 20 ng/ml FGF2, 20 ng/ml EGF, 2% B27 without VA, Glutamax, N2 and  Penicillin/Streptomycin on culture dishes precoated with Matrigel matrix (2%). For knocking out Mettl14 from mouse hypothalamic NPCs, NPCs were incubated with culture medium containing lenti-EGFP-Cre or lenti-EGFP and the medium is refreshed after a 24-hour incubation period.After 72 hours, we isolate EGFP-positive cells using flow cytometry at the Penn Core Facility. 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. Optimizing m6A-modified RNA enrichment via the utilization of polyadenylated RNA fragments from 30µg total RNA with EpiMark N6-Methyladenosine Enrichment Kit (NEB) 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).