GSM6704272: antim6A_bound_1; Mus musculus; RNA-Seq
polyA RNA was isolated from sorted Cxcl12-DsRed+ cells using Dynabeads Oligo-(dT)25 magnetic beads (ThermoFisher Scientific, Cat# 61011) according to manufacturer's instructions. 6% of eluted RNA was saved as input fraction. 1.25 μg of antim6A antibody (Synaptic Systems, Cat#202003) or IgG was pre-bound to Protein A/G magnetic beads (Millipore, Cat#16-663X) in IP buffer (20-mM Tris pH 7.5, 140-mM NaCl, 1% NP-40, 2-mM EDTA) for 1 h. Sample RNA was incubated with antibody-bound Protein A/G beads for 2 hours at 4 ℃. Samples were washed twice in low-salt-wash buffer (10 mM Tris, pH 7.5, 5 mM EDTA), twice with high-salt-wash buffer (20 mM Tris, pH 7.5, 1 M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA), and then twice with RIPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA). All wash solutions for each sample were collected as the “unbound” fraction. RNA was eluted from the beads by incubating with 50 μl 20 mM N6-methyladenosine 5-monophosphate sodium salt (Sigma-Aldrich, Cat# M2780) for 1 hour at 4 ℃. Following ethanol precipitation, input, unbound, and bound fractions was reverse transcribed, amplified, tagmented and indexed following Smart-seq2 method (Picelli et al., 2014)