GSM9631135: Arc Cntrl; Mus musculus; RNA-Seq
RNA was extracted from TRIzol Reagent (Invitrogen) as per the manufacturer's instructions, using 10µg RNAse free Glycogen (Thermo Scientific) as a carrier. Biotinylated RNA enrichment was performed similar to as described in stress granule APEX-seq1 All enrichment buffers were made fresh from RNAse free ingredients whenever possible and all washes occurred on a magnetic rack. Briefly: 10µl of Dynabeads MyOne Streptavidin C1 beads(ThermoFisher) per sample was washed three times with 1mM MgCl2, 0.25% sodium deoxycholate, 1x PBS wash buffer. Beads were then washed once and blocked for 30 minutes in 5x Denhardt's reagent, 150 µg/mL Poly IC, 1mM MgCl2, 1x PBS, 0.25% sodium deoxycholate, 1:100 Ribolock RNAse inhibitor blocking buffer. Block was removed and RNA samples were added along with fresh blocking buffer. Samples were incubated on a slow rocking nutator at RT for 1.5hrs ensuring sample mixing. Beads were washed twice with 6M urea, 0.1% SDS, 1x PBS. Beads were washed once with 2% SDS, 1x PBS. Beads were washed once with 750 mM NaCl, 0.25% sodium deoxycholate, 0.1 % SDS, 1x PBS. Beads were washed once with 150mM NaCl, 0.25% sodium deoxycholate, 0.1 % SDS, 1x PBS. RNA was eluted from the beads with 300uL TRIzol and purified with the Direct-Zol RNA direct-zol RNA microprep kit (Zymogen). RNA was not quantified after pulldown, it was frozen at -80C or immediately processed for first strand cDNA synthesis using the Smartseq v4 mRNA Library prep kit (Takara) with maximum RNA input volume.       .