Illumina HiSeq 2500 paired end sequencing; Single-cell RNA-seq of murine medullary thymic epithelial cells (mTECs)
Single-cell RNA-seq of murine medullary thymic epithelial cells (mTECs)
Thymi were collected from 2 and 4 week-old wild-type C57BL/6 male and female mice. Up to 3 thymi were cleaned of fat and connective tissue, finely minced and pooled together. Thymocytes were flushed by gentle agitation with a magnetic stirrer in RPMI-1640 for 30 min, at 4ºC. Thymic fragments were recovered by settling, and the supernatant discarded. After further dispersion, three additional washes were performed. Fragments were then incubated in 5 mL of 0.125% (w/v) Collagenase D and 0.1% (w/v) DNAse I (both from Roche) in RPMI-1640, at 37ºC for 15 min, with gentle pipetting every 5 min. The supernatant was collected and kept on ice, while the thymic fragments were subject to two further incubations. The remaining fragments were finally resuspended in 5ml of 0.125%(w/v) Collagenase/Dispase (Roche) and 0.1%(w/v) DNaseI in RPMI-1640 for 30 min at 37ºC, with gentle agitation every 15 min. All the collected fractions were pooled, centrifuged at 450x g for 5 min and incubated in 5 mM EDTA, 1% FCS, 0.02% (w/v) NaN3 in PBS (EDTA/FACS buffer) for 10 min at 4ºC. After filtering through a 100μm-strainer, the resulting cell suspension was depleted of hematopoietic cells by Magnetic-Activated Cell Sorting (MACS) using CD45-MicroBeads (Miltenyi Biotec). For sorting, the recovered fraction was blocked using anti-CD16/CD32 (clone 2.4G2, Tonbo) and then stained using anti-CD45-PerCP-Cy5.5 (clone 30-F11, BioLegend), anti-Ly-51-FITC (clone 6C3, BioLegend), anti-CD326(Ep-CAM)-AF647 (clone G8.8, BioLegend), UEA-1-Biotin and Streptavidin-Pacific Blue. Propidium iodide was used as a viability dye. mTECs (PI−CD45−EpCAM+Ly51−UEA-1+) were sorted with a MoFlo™ XDP (Beckman Coulter, Inc.). Single cell capture and processing for the main dataset was performed using the Fluidigm C1 system. The cell suspension obtained from sorting was loaded onto the Fluidigm C1 platform using medium–sized capture chips (10-17μm cells). 1 μl of a 1:400 or 1:2000 dilution of External RNA Control Consortium (ERCC) spike-ins (Ambion, Life Technologies) were included in the lysis buffer. Reverse transcription and cDNA preamplification were performed using the SMARTer Ultra Low RNA kit (Clontech). The cDNA libraries for sequencing were prepared using Nextera XT DNA Sample Preparation Kit (Illumina), according to the protocol supplied by Fluidigm (PN 100-5950 B1). Libraries from 96 single cells were pooled and purified using AMPure XP beads (Beckman Coulter).