GSM6213575: IRE1F/F_rep 2; Mus musculus; RNA-Seq
The mouth of a euthanized mouse was rinsed in 1mL cold PBS containing ProtectRNA (Sigma) and the tongue was excised from the mouse. The mucosa was separated from the submucosa using an adapted version of a previously described method (Dando et al., 2015; Venkatesan, Boggs and Liu, 2016). Briefly, the tongue was injected under the epithelium with 1 mL of an ice-cold solution of 1 mg/mL neutral protease (Worthington), 1 mg/mL collagenase 1 (Worthington) and ProtectRNA (Sigma) in RNase-free PBS. The inflated tongue was immersed in the enzyme solution and incubated at 37°C for 10 mins. The tongue was cut in half lengthways, the mucosa was peeled from each half, rinsed in PBS and then placed in RNAlater (Ambion). The tissue was homogenized with a 1.5 mL tube pestle, followed by treatment with QiaShredder (Qiagen). RNA was extracted from the tissue using an RNeasy Mini Plus kit (Qiagen). Library preparation was performed using QuantSeq 3' mRNA-Seq kit