title: P3OMP21
organism: Mus musculus
description: Protocols: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-12-02T04:02:51Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-08-30T10:10:51Z
attributes.tag: External Id
attributes.value: SAMEA5925656
attributes.tag: INSDC center name
attributes.value: Cancer Research UK Cambridge Institute.
attributes.tag: INSDC first public
attributes.value: 2019-12-02T04:02:51Z
attributes.tag: INSDC last update
attributes.value: 2019-08-30T10:10:51Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8285:P3OMP21
attributes.tag: age
attributes.value: 3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: postnatal
attributes.tag: genotype
attributes.value: OMP-GFP heterozygote
attributes.tag: inferred cell type
attributes.value: olfactory sensory neuron
attributes.tag: organism part
attributes.value: olfactory epithelium
attributes.tag: sample name
attributes.value: E-MTAB-8285:P3OMP21
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: B6-N11