title: Fhit polysome study induced replicate 2
design: Cells were seeded, cultured, and induced as indicated in (Kiss et al., Biochim Biophys Acta. 2017 Jan 14;1860). Cells were then washed twice with PBS, scraped, and pelleted by centrifugation at 2500 xg for 5 min at 4°C. Cell pellets were lysed in 5 pellet volumes of ice cold polysome lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 150 μg/ml CHX, 1% Triton X-100), incubated on ice for 10 mins with mild agitation every 2 minutes. Cellular debris was pelleted by centrifugation (16,100 xg) for 10 mins at 4°C and the supernatants were transferred to fresh RNase-free tubes.  RNA was harvested from 200 ul of the resulting cytoplasmic extract using the Direct Zol kit from Zymo according to their reaction cleanup protocol.  The total RNA was measured for concentration and RNA integrity and then sent to Hudson Alpha Institute for ribosomal RNA reduction, barcoded library preparation, and sequencing.
identifiers.namespace: SUB3200101
identifiers.id: FHIT_D1_IND_R2_RNA