title: Illumina HiSeq 2500 paired end sequencing; Single cell RNA-sequencing of induced Neural Border Stem Cells
design: Single cell RNA-sequencing of induced Neural Border Stem Cells
library_construction_protocol: Adult dermal fibroblasts (ADFs) were derived from the skin biopsies of a healthy male donor (Age 24) as described in Meyer et al. 2015 ( Meyer, S., Wörsdörfer, P., Günther, K., Thier, M., Edenhofer, F., 2015. Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells. J Vis Exp e52831–e52831. doi:10.3791/52831). ADFs were expanded until passage 4 and frozen in 90% Serum/ 10% DMSO prior to use. All culture were grown at 37°C in 5% CO2 and 20% O2. Peripheral blood mononuclear cells (PBMCs) were derived from a healthy male donors (Age 26) and isolated using the Ficoll gradient procedure. PBMCs were used either directly or frozen in 90% Serum Replacement/ 10% DMSO as previously described (for details see Sommer, A.G., Rozelle, S.S., Sullivan, S., Mills, J.A., Park, S.-M., Smith, B.W., Iyer, A.M., French, D.L., Kotton, D.N., Gadue, P., Murphy, G.J., Mostoslavsky, G., 2012. Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector. J Vis Exp e4327–e4327. doi:10.3791/4327). Cells were derived from informed consent from all donors and handled in accordance with Ethics Committee II of Heidelberg University approval no. 2009-350N-MA. To reprogramme cells we used the pHAGE2-Vector (pHAGE2-TetO-BKSZ and pHAGE2-TetO-BKSZ-flox) in combination with the transactivator m2RTTA (Addgene plasmid #20342) for the overexpression of the transgene cassette. ADF2 were transduced with pHAGE2-TetO-BKSZ + m2RTTA; PBMC1 were transduced with pHAGE2-TetO-BKSZ-flox and m2RTTA. Induced Neural Border Stem cells were harvested by treatment with Accutase and 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. NBSC maintenance: NBSC maintenance medium is composed of DMEM/ F-12 Glutamax (for iNBSCs) or 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. NBSCs were cultured in NBSC maintenance medium on a layer of inactivated mouse embryonic fibroblasts (feeder) at 37°C in 5% O2 and 5% CO2. iNBSC lines were routinely split after 4-5 days by treatment with Accutase (Life Technologies) and transferred onto fresh feeders. Conversion into iNBSCs: Lentiviral supernatants containing BKSZ-flox and m2RTTA were mixed in a ratio of 2:1, supplemented with 5 µg/ml polybrene (Sigma) and used freshly for transduction of 8x10**5/ 6 Well primary ADF and pancreas fibroblasts. For reprogramming of PBMCs the lentiviral supernatant was concentrated via ultracentrifugation and PBMCs were transduced in QBSF-60 Stem Cell Medium (Quality Biological) containing 50 μg/ml Ascorbic Acid (Sigma), 50 ng/ml SCF (R&D Systems), 10 ng/ml IL-3 (R&D Systems), 2 U/ml EPO (R&D Systems), 40 ng/ml IGF-1 (Peprotech), 1 μM Dexamethasone (Sigma) and 5 µg/ml polybrene (for details see {Sommer:2012db}). The other day 2x10**5 transduced PBMCs were transferred onto one well of a 6 Well plate, coated with inactivated MEFs and reprogramming was initiated one day thereafter.For reprogramming transduced cells were cultured in DMEM/F-12 Glutamax (Life Technologies) containing 64 µg/ml L-Ascorbic acid 2-phosphate (LAAP, Sigma), ITS-X (1:100, Life Technologies), NEAA (1:100, Life Technologies), 2% FCS, 8% Serum Replacement (Life Technologies) supplemented with 4 µM Chir99021 (Sigma), 5 µM Alk5 Inhibitor II (Enzolifesciences), 0.5 µM Purmorphamine (Sigma), 5µM Tranylcypromine (Sigma) and 1µg/ml Doxycycline (Sigma) and incubated at 37°C in 5% O2 and 5% CO2. During reprogramming medium was changed every other day. When first colonies became visible or latest after 19 days, medium was changed to NBSC maintenance medium. 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.192134.LR.29147
attributes.tag: Experimental Factor: progenitor cell type
attributes.value: fibroblast of dermis