title: GSM8974311: Human SmB Sm-RIP, stringent washing 1 (hbs1); Homo sapiens; RIP-Seq
library_construction_protocol: Cytoplasmic cell extracts were generated by scraping and collecting NSC-34 or S3-iNPC cells in cold phosphate buffered saline containing only monovalent cations. Cells were pelleted by centrifugation 400 x g for 4 min. Cells were resuspended in digitoxin lysis buffer (20 mM Tris pH 8, 50 mM KCl, 0.5 MgCl2, 1x eComplete, 200 units/mL RNase-OUT, 50 ug/mL digitonin, in DEPC-treated water) and incubated on ice for a few minutes. Lysates were passaged 6 times through a 27 gauge needle with syringe then centrifuged at 1,500 x g for 1 minute at 4 °C. Supernatant was measured and transferred to a new microfuge tube and supplemented with NP-40 to achieve 0.01% final concentration. Lysates were then centrifuged at 4,000 x g for 15 minutes at 4 °C. Supernatants were then aliquoted into microfuge tubes and frozen in liquid nitrogen. Three replicates of the four conditions for both species combinations were used to generate sequencing libraries, using 50 ng of captured RNA as input for the Takara SMARTer Stranded Total RNA-Seq Kit v3-Pico Input Mammalian (Takara 634451) or Takara SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara 634411).