Mosquito hts

SP100C crystals were grown in SWISSCI 3 Lens crystallisation sitting‐drop plates at 4 °C by mixing 50–100 nL of 10 mg mL⁻¹ protein solution in a 1:1 ratio with 50–100 nL reservoir solution consisting of 0.1 m MES pH 7.0, 20–30 % (w/v) PEG 20000 and placing the drops over 20 μL reservoir solution. Crystals appeared in 6–7 days. Crystal soaking was performed by liquid droplet transfer using a TTPLabtech Mosquito® HTS. Ethylene glycol was added for cryoprotection using the Mosquito® to a final concentration of 25 % (v/v) (calculated from the initial drop volume). The SP100C crystals diffracted to 1.6–2.0 Å resolution in space group C2, with typical unit‐cell parameters a=127.4 Å, b=45.4 Å, c=84.0 Å, β=102.0° and with one SP100C molecule in the asymmetric unit.
X‐ray diffraction data were collected on beamline I04‐1 at Diamond Light Source and were processed using the Diamond autoprocessing pipeline, which uses xia2,48 DIALS,49 XDS,50 POINTLESS,51 and CCP4.52 Electron‐density maps were generated using XChemExplorer45 via DIMPLE.53 Ligand restraints were generated with AceDRG54and iterative refinement and manual model correction was performed using REFMAC55 and Coot,56 respectively.

For single-cell sequencing of the tissue, mouse thyroid was dissociated with collagenase I (Sigma-Aldrich) as described above and subsequently resuspended in TrypLE Express (GIBCO) preheated to 37 °C and dissociated under repeated pipetting. For single-cell sequencing of TFCOs, organoid droplets were incubated in Cell Recovery solution (354253, Corning) for 30 min in order to dissolve the BME. Then, organoids were pelleted and resuspended in TrypLE Express (GIBCO) preheated to 37 °C and dissociated under repeated pipetting. When the gland and the organoids were fully dissociated into single cells, samples were pelleted, washed, resuspended in fluorescence-activated cell sorting (FACS) buffer (advanced DMEM/F12, 10 μM Y-27632, and DAPI) and strained (35 μm).
DAPI-negative cells were immediately sorted into 384-well plates containing External RNA Controls Consortium (ERCC) spike-ins (Agilent), reverse transcription (RT) primers, and deoxynucleotide triphosphates (dNTPs) (Promega) using a FACSFusion (BD Biosciences). Plates were prepared using Mosquito HTS (TTP Labtech). Single-cell RNA-sequencing libraries were prepared following the SORT-seq protocol (26 (link)), which is based on the CEL-seq2 method (53 (link)). Extensive explanation of the protocol can be found in SI Appendix.

Not all 7500 sorted cells were sequenced; rather, to improve coverage at the same cost, around 2000 cells were cherry picked for sequencing. With the results of the cDNA quantification and the virus and ACTB qPCR at hand, cells were selected such that they cover the largest possible set of conditions. For instance, in a plate with infected cells we ensured that both qPCR negative cells (ACTB but no virus), cells with little virus, and cells with a high amount of vRNA were all represented in the sequencing data. The selection was designed in a semi-automatic way via JavaScript and Python scripts and implemented on TTPLabtech Mosquito HTS and X1 HV robotic platforms. At the same time as cherry picking, the cDNA from each cell was also diluted to around 0.4 ng/ul for Tn5 endonuclease library prep. Although this concentration is slightly higher than usual or this type of libraries, the cDNA was not purified so that a certain fraction of the DNA is residual short oligos from previous reactions, which is most likely too short to end up on the sequencer.