GSM8851178: cells for doublet study, date 202302 (batch 3); Mus musculus; RNA-Seq
After seeding into U40 nanowell slides, CD8+ T cells, DCs, and doublets were picked by the 30 µm glass capillary under the automatic picking mode of CellCelector (Sartorius AG, Niedersachsen, Germany). Three channels (i.e. brightfield, FITC for CD8+ T cells labelled with CellTrace CFSE, and TRITC for DCs labelled with CellTrace Yellow) were selected for scanning to validate cell type. Captured cells with 0.1 uL DNase/RNase-Free Water were dispensed into a 384-well Frame-Star plate containing 1.1 μL lysis buffer. The lysis plate was cooled down to 4 °C at the rack tray during cell capture and was stored at −80 °C for subsequent procedures. The library was prepared following Smart-seq2 protocol 58. Briefly, cells were picked into 384 well lysis plates followed by reverse transcription, template switching and 23 cycles of PCR to generate and amplify the cDNA. MANTIS automated liquid handler (Tecan, Zürich, Switzerland) was used during the process. cDNA was normalised to 0.1-1 ng/µl according to the qualification by Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, Waltham MA, USA) via automated liquid handling robot Mosquito (SPT Labtech, Melbourn, Cambridgeshire, UK). TruePrep DNA Library Prep Kit V2 (Vazyme #TD503, Nanjing, China) with 15 cycles of amplification were used for tagmenting immune cells. Purification was conducted to remove primer by Agencourt Ampure XP (Beckman Coulter, Brea CA, USA) magnetic beads at a ratio of 0.7x two times. Libraries were quantified using Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany).