GSM6217297: E13.5 neocortex, IgG; Mus musculus; RIP-Seq
Mouse E13.5 cerebral cortices were dissected and enzymatically dissociated to a single-cell suspension. Cortical cells were washed in ice-cold DPBS and lysed in RIP lysis buffer A (10mM Tris pH7.4, 140mM NaCl, 1.5mM MgCl2, 0.5% NP-40, 1mM dithiothreitol, SUPERaseIn RNase inhibitor, proteinase inhibitor cocktail). Lysates were incubated on ice for 5 minutes, and then centrifuged 3 minutes at 4℃ and 1000g. The supernatant was collected as cytoplasmic fractions and used as input or for RIP with anti-eIF4E antibody or control immunoglobulin G (rabbit) incubating for 2 hours at 4℃ with gentle rotation. Then washing off unbound material from magnetic beads for 4 times. Isolate coprecipitated RNAs were extracted using TRIzol reagent (Invitrogen) and processed according to the manufacturer's instruction. cDNA libraries from pulled-down RNA and input RNA were prepared using standard Smart-seq2 protocols for Illumina novaseq 6000.