Illumina HiSeq 2500 paired end sequencing; TOMO-seq along lateral axis, dorsal-ventral axis and anterior-posterior axis of mouse main olfactory epithelium
TOMO-seq along lateral axis, dorsal-ventral axis and anterior-posterior axis of mouse main olfactory epithelium
All mice used were C57BL/6J (The Jackson Laboratory, Stock # 00664), 8-9 weeks old and group-housed. The whole olfactory mucosa (WOM) of 9 mice was carefully dissected, and all the surrounding tissue (including glands and bone) removed. The WOMs were then embedded in OCT (Tissue Tek), immediately frozen in dry-ice and kept at -80ºC.   Each WOM was then cryosectioned along each of the 3 cartesian axes: dorsal-ventral (D-V, N=3), anterior posterior (A-P, N=3), or lateral-medial-lateral (L-M-L, N=3). Every second cryosections (35 µM) was collected into 1.5 mL eppendorf tubes containing 350 µL RLT Plus Buffer (Qiagen) supplemented with 1% 2-mercaptoethanol, immediately frozen in dry-ice and kept at -80C until extraction. RNA was extracted using the RNeasy Plus Micro Kit (Qiagen), together with genomic DNA eliminator column and a 30 minute incubation with DNAse I (Qiagen). Reverse transcription and cDNA pre-amplification were performed using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech/Takara). cDNA was harvested and quantified with the Bioanalyzer DNA High-Sensitivity kit (Agilent Technologies). Libraries were prepared using the Nextera XT DNA Sample Preparation Kit and the Nextera Index Kit (Illumina).