NextSeq 550 paired end sequencing; Targeted scRNAseq of control and SPP1 stimulated human whole blood
Targeted scRNAseq of control and SPP1 stimulated human whole blood
Blood samples from (n=3) healthy donors were collected by venipuncture into heparin collection tubes. RBC lysis was performed for 5 mins at room temperature incubation with ACK Lysing Buffer (A1049201, ThermoFischer Scientific) before leukocyte collection by centrifugation. Leukocytes from each donor were plated at a density of 5 × 106 per well in a 24-well cell-culture plate in 0.5 ml of RPMI 1640 compete medium containing SPP1 (PeproTech) at concentrations of 0, 50 and 200ng ml–1. After 18 hours, cells were de-attached using Accutase solution (no. A6964, Merck) according to the manufacturer's protocol. Cells were transferred to U-bottom 96-well plates and harvested by centrifugation at 200 g for 4 min at 4 °C. Cells from each donor/condition were labelled with unique Tags using a Single-Cell Multiplexing Kit (BD, no. 633781) for 15 min at room temperature according to the manufacturer protocol (BD). Cells from each donor/condition were labelled with unique tags using a Single-Cell Multiplexing Kit (BD, no. 633781) for 15 min at room temperature according to the manufacturer protocol (BD). The following sample tags were used:Sample Tag 1— ATTCAAGGGCAGCCGCGTCACGATTGGATACGACTGTTGGACCGG,Sample Tag 2—TGGATGGGATAAGTGCGTGATGGACCGAAGGGACCTCGTGGCCGG,Sample Tag 3—CGGCTCGTGCTGCGTCGTCTCAAGTCCAGAAACTCCGTGTATCCT,Sample Tag 4—ATTGGGAGGCTTTCGTACCGCTGCCGCCACCAGGTGATACCCGCT,Sample Tag 5—CTCCCTGGTGTTCAATACCCGATGTGGTGGGCAGAATGTGGCTGG,Sample Tag 6—TTACCCGCAGGAAGACGTATACCCCTCGTGCCAGGCGACCAATGC,Sample Tag 7—TGTCTACGTCGGACCGCAAGAAGTGAGTCAGAGGCTGCACGCTGT,Sample Tag 8—CCCCACCAGGTTGCTTTGTCGGACGAGCCCGCACAGCGCTAGGAT,Sample Tag 9—GTGATCCGCGCAGGCACACATACCGACTCAGATGGGTTGTCCAGG.  Cells were then washed three times with PBS, with centrifugation steps (200g for 4 min at 4 °C), after which the tagged culture variants were pooled and loaded onto the scRNA-seq BD Rhapsody Cartridge using the BD Rhapsody Cartridge Reagent Kit (no. 633731) according to the manufacturer's protocol. Single-cell cDNA was prepared using the BD Rhapsody cDNA Kit (no. 633773). This was followed by single-cell mRNA and Tag library preparation using BD Rhapsody Targeted mRNA and the Tag Amplification Kit (no. 633774) and primers for the BD Rhapsody Immune Response Panel (399 genes; no. 633750).   Cells from each donor/condition were labelled with unique tags using a Single-Cell Multiplexing Kit (BD, no. 633781) for 15 min at room temperature according to the manufacturer protocol (BD). The following sample tags were used:Sample Tag 1—