GSM2664777: E12.5_E21 [single cell RNA-seq]; Mus musculus; RNA-Seq
Timed-pregnant mice were sacrificed by deep anesthesia followed by cervical dislocation. Dissection of embryos and adrenal anlages was carried out using a stereotactic microscope equipped with a fluorescent light source. Biological materials were transferred into DMEM/F12 medium with 0.05% Trypsin/ 0.02% EDTA (Sigma) and incubated at 37⁰ C for 5 min. Single cells were isolated by gentle trituration using P1000 tips and then added cold 10% FCS to stop the enzymatic reaction. After a double wash procedure using PBS with RNAse inhibitor, cells were filtered through a 40 uM nylon cell strainer and collected into a FACS tube on ice. Samples remained on ice throughout the process and total processing time from adrenal anlages isolation to sorting was limited to 1 hour. Dissection of Wnt1-Cre/Tomato+ sympatho-adrenal anlages at E12.5 and E13.5 stages was carried out using a stereomicroscope equipped with a fluorescent light source. The dissected fragments were transferred into DMEM/F12 medium with 0.05% Trypsin/ 0.02% EDTA (Sigma) and incubated at 37⁰ C for 5 min. Single cells were released by gentle trituration using P1000 tips, and then ice-cold 10% FCS was added to stop the enzymatic reaction. After a double wash procedure using PBS with RNAse inhibitor, cells were filtered through a 40 uM nylon cell strainer and collected into a FACS tube on ice. After that the samples remained on ice or were kept at +4C throughout the rest of the processing that included single cell FACS sorting of Tomato+ cells into 384-well plates with pre-filled lysis buffer.The sequencing libraries were generated using the published protocol for Smartseq2 sequencing (Picelli et al., Nature Protocols 2014). Resulting libraries were sequenced on the Illumina® HiSeqTM 2000 sequencing platform. The extended analysis is published in Furlan, Dyachuk and Kastriti et al., Science 2016".
GEO
GSM2664777
GEO Accession
GSM2664777