GSM6280561: lncRNA Capture-Seq synapse biol rep2; Mus musculus; ncRNA-Seq
Preparation of synaptosomes was carried out as previously described. Briefly, tissues were homogenized in homogenizing buffer with 10-14 strokes using a Teflon-glass tissue grinder. The homogenate was centrifuged at 1000 x g for 10 mins at 4°C. The supernatant was directly applied onto a discontinuous Percoll gradient ranging from 0% up to 23% Percoll and centrifuged at 31,000 x g for 5 mins to isolate the synaptosome fraction. RNA was extracted according to the manufacturer's instructions. The concentration of RNA was measured using a nanophotometer (IMPLEN) or Qubit fluorometer (Thermo Fisher Scientific). Synaptosomes were collected and total RNA was isolated as described above. cDNA libraries were generated using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's protocol. A custom-designed probe panel (ROCHE) targeting 28,228 mouse lncRNAs18 was used to capture amplified cDNA. The capture procedure was performed using the SeqCap EZ Hybridization and Wash Kit (ROCHE) and SeqCap EZ Accessory kit (ROCHE) according to the manufacturer's instructions. Captured libraries were sequenced on an Illumina HiSeq 4000 platform with 150-bp paired-end reads (GENEWIZ) lncRNA capture sequencing