title: Illumina HiSeq 2500 paired end sequencing; Single cell RNA-sequencing of primary Neural Border Stem Cells
design: Single cell RNA-sequencing of primary Neural Border Stem Cells
library_construction_protocol: Mouse pNBSCs were derived from a mixed 129X1/SvJ background and are homozygous for Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo (see also: https://www.jax.org/strain/007576). Induced Neural Border Stem cells were harvested by treatment with Accutase and washed twice with unsupplemented DMEM/ F12. Subsequently iNBSCs were stained for CD133/2 (293C3)-FITC (Miltenyi Biotec) and anti-CXCR4 (12G5)-PeCy5 (BioLegend) for 30 minutes at 4°C in basal medium and washed twice with DMEM/ F12. 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 of 1: 1 000 000 diluted stock of ERCC spike-ins (ThermoFisher)) in separate 96 wells and flash-frozen in liquid N2 until further processing. NBSC maintenance medium is composed of Adv. DMEM/ F12 Glutamax (for pNBSCs) and Neurobasal Medium (1:1) containing 64 µg/ml LAAP, N2 Supplement (1:100, Life Technologies), B27 without RA (1:50, Life Technologies), 18 µg/ml Albumax I (Life Technologies) and Glutamax (1:200, Life Technologies), referred to as 'basal medium', and 4 µM Chir99028, 5µM Alk5 Inhibitor II and 0.5 µM Purmorphamine. Once visible, distinct colonies were manually picked and cultured in NBSC maintenance medium on a layer of inactivated mouse embryonic fibroblasts (feeder) at 37°C in 5% O2 and 5% CO2. 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 decribed 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.215429.LR.32701