GSM5512992: Sciatic nerve ribotag (SN_ribotag_3_TGAGGTT); Mus musculus; RNA-Seq
The protocol was modified based on Sanz et al. Specifically, we generated Mpz-Cre:Ribo-Tag mice that, upon the expression of Mpz, cells would express HA-tagged ribosomal protein RPL22. The mRNA of Mpz-expressing cells from the nerve was then isolated by immunoprecipitation of polyribosomes with anti-HA and subjected to RNA-seq. Both direct and indirect pull down approaches were tested and indirect pull down was selected because of higher yields. Both sciatic and vagus nerves were dissected from Mpz-Cre:RiboTag mice and immediately placed into PBS with RNaseIn and SuperaseIn on ice. The epineurium of sciatic nerve was also carefully removed by forceps before placing the remaining nerves into PBS. Upon dissection, the nerves were transferred to dissociation media with TL liberase, RNaseIn and SuperaseIn for enzymatic dissociation for 15min at 37°C. The cells were pelleted by spinning at 1500rpm for 5 min, homogenized using Dounce homogenizer in Polysome buffer and then spin at 10,000g for 10min. Supernatant was then collected, with HA antibody-coupled magnetic beads added to the cleared supernatant. After an overnight incubation at 4°C, the magnetic beads that contained immunoadsorbed polysomes were washed with a high salt buffer. mRNA was then extracted using the Qiagen RNA extraction kit according to manufacturer's protocol. Samples were prepared according to Illumina library kit manufacturer's protocol, indexed, pooled, and sequenced on an Illumina HiSeq.