Illumina NovaSeq 6000 paired end sequencing; Charting the age-altered thymic epithelial cell differentiation by lineage tracing from a beta 5-t expressing TEC progenitor
Charting the age-altered thymic epithelial cell differentiation by lineage tracing from a beta 5-t expressing TEC progenitor
One-week old ﻿3xtgβ5t mice were treated with a single i.p. injection of 0.004mg of Doxycycline (Sigma) diluted in Hank's Balanced Salt Solution (Life Technologies), whereas older mice (four-week and sixteen-week old) were treated with ﻿two i.p. injections of Doxycycline (2mg, each) on two consecutive days during which they were also exposed to drinking water supplemented with the drug ﻿(2 mg/mL in sucrose (5% w/v)). Single thymic epithelial cell suspensions were obtained by enzymatic digestion using Liberase, Papain and DNase in PBS. Prior to FAC-sorting, TEC were enriched for EpCAM-positivity using a magnetic cell separator, as described above. Enriched cells were subsequently stained for the indicated cell surface antigens EpCAM, Ly51, CD80, MHCII & UEA1 in conjunction with TotalSeq-A oligonucleotide-conjugated antibodies against mouse MHCI and CD45 to allow barcoding and pooling of different TEC subpopulations and subsequently sorted into 4 subpopulations: ZsGreen+ cTEC, ZsGreen- cTEC, ZsGreen+ mTEC, and ZsGreen- mTEC. After sorting, the cell viability and concentration of each of the cell samples collected was measured using a Nexcelom Bioscience Cellometer K2 Fluorescent Viability Cell Counter.  Equal cell numbers were pooled from each of the samples, and a total of 30000 cells were loaded per well onto a Chromium Single Cell B Chip (10x Genomics) coupled with the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 and Chromium i7 Multiplex Kit (10x Genomics) for library preparation, according to the manufacturer's instructions. In short, the cell suspension was mixed with the GEM Retrotranscription Master Mix and loaded onto well number 1 on the Chromium Chip B (10x Genomics). Wells 2 and 3 were loaded with the appropriate volumes of gel beads and partitioning oil, respectively, after which the Chromium Controller (10x Genomics) was used to generate nanoliter-scale Gel Beads-in-emulsion (GEMs) containing the single cells to be analysed. The fact that cell samples containing 6 different hashtag antibodies were pooled together allowed us to overload the 10x wells with 30000 cells per well, aiming for a recovery of approximately 12000 single cells (40%) per well, and being able to overcome the increase in doublet rate by subsequently eliminating any cell barcode containing more than one single hashtag sequence from further analysis. Incubation of the GEM suspension results in the simultaneous production of barcoded full-length cDNA from poly-adenylated mRNA as well as barcoded DNA from the cell surface protein-bound TotalSeqA antibodies inside of each individual GEM. Fragmentation of the GEMs allowed the recovery and clean-up of the pooled fractions using silane magnetic beads. Recovered DNA was then amplified, and cDNA products were separated from the Antibody-Derived Tags (ADT) and Hashtag oligonucleotides (HTO) by size selection.  The amplified full-length cDNA generated from polyadenylated mRNA were enzymatically fragmented and size selection was used to optimise amplicon size for the generation of 3' libraries. Library construction was achieved by adding P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) via End Repair, A-tailing, Adaptor Ligation, and PCR. Separately, ADT and HTO library generation was achieved through the addition of P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) by PCR.