GSM4220535: SN-IgG-3; Mus musculus; RNA-Seq
For enriched axoplasm extraction Sciatic nerves (SN) or Dorsal root (DR) tissues were freshly dissected and collected in Nuclear Transport buffer (TB) at a ratio of 1 SN or 3DR / 50 ml buffer (TB: 20 mM Hepes, 110 mM KAc, 5 mM MgAc, pH 7.4 supplemented with Complete protease inhibitor EDTA free, 1/50 dilution of 1 tablet reconstituted in 1ml water, as recommended by the manufacturer (Roche 1187358000), phosphatase inhibitor cocktail 2 (1/1000, Sigma 5726), phosphatase inhibitor cocktail 3 (1/1000, Sigma P0044) and RNAse inhibitors (200U/ml, RNAseIn, Promega N251B)). Tissue was manually grinded with a micropestle in Eppendorf tube until it lost its fibrous consistency. Lysates were centrifuged 10,000 g for 10 minutes at 4°C and pellet was discarded. approximately 200ug of axoplasm/protein lysates were incubated with 10ug antibody for 3 hours, then supplemented with 100ml Protein G magnetic beads (Dynabeads, Thermo Fisher Scientific 10004D), pre-blocked with salmon sperm DNA (10ml DNA pwe 100ml beads for 1 hour 4°C), for additional 1 hour. Incubation buffer was adjusted to final 0.1% NP40 to avoid beads aggregation. All incubation steps were done at 4°C with overhead rotation. Beads were washed with increasing NP40 concentration as follows: TB-0.1% NP40 for 3-5 minutes, TB-0.5% NP40 3-5 minutes, TB-1% NP40 1 minute, TB- no detergent 3-5 minutes, then transferred to a clean Eppendorf tube in TB without detergent. RNA was extracted from the beads using Qiagen RNeasy micro kit (Qiagen, 74004) as following the manufacturer protocol. Briefly, samples were lysed in ± 300l RLT buffer supplemented with 400mM DTT by repeated vortexing (5 times, 4 seconds on low speed). Supernatants were kept and supplemented with 70% ethanol to reach 150% of recovered volume. Samples were loaded on columns and manufacturer protocol was followed. DNase treatment was conducted on-columns for 10 minutes. Elution of RNA from the columns was done using 13l DNase/RNase free water. SMART-Sq v4 RNA Ultra Low Input (10pg) + Nextera XT library
GEO
GSM4220535
GEO Accession
GSM4220535