title: Illumina HiSeq 2500 paired end sequencing; Single cell RNA-sequencing of E8.5 stage, embryonic neural tissue from mouse
design: Single cell RNA-sequencing of E8.5 stage, embryonic neural tissue from mouse
library_construction_protocol: Male and female C57BL6/J mice were paired and checked for vaginal plug the following morning. Positive plug test was considered 0.5 days post coitum. Embryos were collected at day 8.5 post coitum and optic and non-neural tissues were removed mechanically. Neural tissue was digested with Accutase and the resulting single cell suspension was washed twice with unsupplemented DMEM/ F12. After surface staining for 30 minutes at 4°C, cell sorting was carried out on a BD FACSAria™ III sorter (Becton Dickinson, San Jose, CA). The following sort parameters were used: 100µm nozzle; up to 800 events/second. Single cells were sorted into lysis buffer (2.86µl H2O, 1µl dT30VN (10µM), 1µl dNTPs, 0.1µl RNAse inhibitor, 0.04µl Triton X-100, 0.081µl ERCC spike-ins) in separate 96 wells and flash-frozen in liquid N2 until further processing. As cells were sorted directly into lysis buffer there was no visual pre-sequencing quality check. Inferred cell type annotation was determined by a classification algorithm (Ben-hur, 2008), which was trained on a dataset of whole E8.25 mouse embryos, defining numerous cell types by single-cell RNA-sequencing analysis (Ibarra-Soria et al., 2018). Single-cell RNA sequencing was performed following the Smart-seq2 protocol with adaptation as outlined below (Picelli et al. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nature Protocols, 9(1), 171–181. http://doi.org/10.1038/nprot.2014.006).Plates containing single cells in lysis buffer were thawed on ice, incubated at 72°C for 3 minutes and reverse transcribed using the SmartScribe RT Kit (Takara Bio Clontech): 2µl Smart FS buffer, 0.5µl DTT, 0.1µl 100µM TSO, 1µl Smarc Scribe, 1.4µl H2O per cell. PCR preamplification and cDNA purification was performed as described in Picelli et al., applying 19 amplification cycles. For tagmentation 0.15-0.2 ng/µl cDNA was used per reaction: 1.25µl diluted sample, 2.5µl Tagment DNA buffer, and 1.25µl Amplification Tagment Mix. The reaction was neutralized with 1.25µL NT buffer.  The adapter ligation was performed with 5µl of the neutralized tagmentation reaction, 3.75µl of Nextera PCR master mix and 1.25µl of Index-Primer 1 and Index-Primer 2 and amplified as described in the Smart-seq2 protocol. Finally,  192 reactions were were pooled (1µl per reaction) and purified according to the Smart-seq2 protocol.
attributes.tag: Experimental Factor: single cell identifier
attributes.value: AS.215255.LR.32701