HiSeq X Ten paired end sequencing; Single-cell RNA-sequencing of OMP-GFP mouse olfactory sensory neurons
Single-cell RNA-sequencing of OMP-GFP mouse olfactory sensory neurons
Whole olfactory mucosa was dissected from heterozygous postnatal day 3 OMP-GFP (B6;129P2-Omptm3Mom/MomJ, The Jackson Laboratory, Stock # 006667) mice that were backcrossed 10 times with C57BL/6 animals. Tissue was minced and then enzymatically digested in HBSS without Ca2+ and Mg2+, supplemented with 44 U/mL dispase (Invitrogen), 1000 U/mL collagenase type II (Invitrogen) and 10 mg/mL DNaseI (Roche), for 15–20 min at 37°C with agitation. Digested tissue was centrifuged at 0.4 x 1000 rcf for 5 min and washed twice in HBSS without Ca2+ and Mg2+. The dissociated cell suspension was passed through a series of filters: 100 µm (Falcon), 70 µm (Flacon) and 20 µm cell strainers (pluriSelect). The final dissociated cells were resuspended in 0.5% BSA in PBS without Ca2+ and Mg2+. Single cells were isolated using a Nikon Narishige microinjection setup under a fluorescence Nikon TE300 microscope. GFP fluorescence was verified on screen using NIS Elements v4.5 software (Nikon) and only GFP-positive cells were selected. Isolated cells were washed twice and then pipetted into a 0.2 mL tube, which was immediately frozen on dry ice. Single cell cDNA libraries were prepared using SMART-Seq v4 (Clontech) according to the manufacturer's recommendations. Briefly, single cells were lysed and reverse transcription was performed with SMART-Seq v4 Oligonucleotide and SMARTScribe Reverse Transcriptase to generate nearly full length cDNA libraries. Libraries were purified twice with AMPure XP system (Beckman Coulter) to minimize primer dimers. The size of the libraries was checked on high‐sensitivity DNA chips (Agilent); cells with abundant short cDNA (average size < 1.3 kb) were discarded. Purified cDNAs were used to construct libraries for sequencing using the Nextera XT DNA Library Preparation Kit (Illumina) according to manufacturer's recommendations. Briefly, cDNAs were fragmented to ~300 bp, followed by PCR amplification with index primers. Libraries were purified with AMPure XP system (Beckman Coulter), normalised and pooled with different i7 indexes.