title: NextSeq 500 paired end sequencing; The role of distinct synovial tissue macrophage populations in shaping synovial tissue microenvironment
design: The role of distinct synovial tissue macrophage populations in shaping synovial tissue microenvironment
library_construction_protocol: Co-culture of distinct STM populations with synovial fibroblasts.  Synovial tissue biopsies from RA patients (n=6) were digested as described above. Synovial fibroblasts were identified  by Thy and PDPN, and STMs were identified by MerTK and CD206, and MerTK/CD206pos, MerTK/CD206neg STMs, and FLS (PDPNpos and PDPN/Thy1pos) were FACS-sorted into Eppendorf tubes containing 0.5ml of complete RPMI1640. FLS were seeded into T25 culture flask (Merck) with complete RPMI1640. After reaching confluence (~1 week) they were harvested and seeded into 96-well plates at 3000 cells/well. After 48h, sorted MerTK/CD206pos or MerTK/CD206neg STMs from RA patients (n=5 patients with active RA and n=5 RA patients in sustained clinical and imaging remission in 5 pairs undergoing US-guided minimally invasive synovial tissue biopsy on the same day) were added to FLS at 3000 cells/well in 3 technical replicates. FLS cultures without STMs provided a negative control. As a positive inflammatory control, FLS co-cultured with MerTK/CD206neg sorted  from RA patients with active RA and pre-treated with LPS (10 ng/ml) for 1 h were used. LPS pre-treated MerTK/CD206neg  were extensively washed before being added to FLS (3 times). After 48h, the phenotype of FLS in co-cultures were evaluated using scRNAseq (BD Rhapsody system). To test the role of MerTK in STM-FLS interactions, in some experimental variants MerTK/CD206pos  STMs were pre-treated with MerTK inhibitor (250 nM, UNC1062). The cells from each co-culture variant were labelled with unique Taqs using Single-Cell Multiplexing Kit (Cat. No. 633781) for 15min at room temperature.  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 Sample Tag 10—GCAGCCGGCGTCGTACGAGGCACAGCGGAGACTAGATGAGGCCCC Sample Tag 11—CGCGTCCAATTTCCGAAGCCCCGCCCTAGGAGTTCCCCTGCGTGC Sample Tag 12—GCCCATTCATTGCACCCGCCAGTGATCGACCCTAGTGGAGCTAAG. Cells were then washed 3x with PBS with centrifugation steps (200g for 4 min in 4ºC). After which the Tagged co-culture variants were pooled and stained with Fixable Viability Dye eFluorTM 780 (eBioscience) as described above. 50x104 live cells were sorted and immediately loaded onto the scRNAseq BD Rhapsody™ Cartridge using BD Rhapsody™ Cartridge Reagent Kit (Cat. No. 633731) according to the manufacturer's protocol.  Single cell cDNA was prepared using BD Rhapsody™ cDNA Kit (Cat. No. 633773). This was followed by single cell mRNA and Tag library preparation using BD Rhapsody Targeted mRNA and Tag Amplification Kit (Cat. No. 633774), and primers for BD Rhapsody™ Immune Response Panel (399 genes; 633750) and custom-made panel that included additional genes expressed by FLS that were not represented in the Human Immune Panel (46 genes).   The cells from each co-culture variant were labelled with unique Taqs using Single-Cell Multiplexing Kit (Cat. No. 633781) for 15min at room temperature.  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 Sample Tag 10—GCAGCCGGCGTCGTACGAGGCACAGCGGAGACTAGATGAGGCCCC Sample Tag 11—CGCGTCCAATTTCCGAAGCCCCGCCCTAGGAGTTCCCCTGCGTGC Sample Tag 12—GCCCATTCATTGCACCCGCCAGTGATCGACCCTAGTGGAGCTAAG. Cells were then washed 3x with PBS with centrifugation steps (200g for 4 min in 4ºC). After which the Tagged co-culture variants were pooled and stained with Fixable Viability Dye eFluorTM 780 (eBioscience) as described above. 50x104 live cells were sorted and immediately loaded onto the scRNAseq BD Rhapsody™ Cartridge using BD Rhapsody™ Cartridge Reagent Kit (Cat. No. 633731) according to the manufacturer's protocol.  Single cell cDNA was prepared using BD Rhapsody™ cDNA Kit (Cat. No. 633773). This was followed by single cell mRNA and Tag library preparation using BD Rhapsody Targeted mRNA and Tag Amplification Kit (Cat. No. 633774), and primers for BD Rhapsody™ Immune Response Panel (399 genes; 633750) and custom-made panel that included additional genes expressed by FLS that were not represented in the Human Immune Panel (46 genes).
attributes.tag: Experimental Factor: growth condition
attributes.value: co-cultured with MerTK/CD206neg macrophages
attributes.tag: Experimental Factor: stimulus
attributes.value: macrophages pre-treated with lipopolysaccharide
attributes.tag: Experimental Factor: single cell identifier
attributes.value: GCCCATTCATTGCACCCGCCAGTGATCGACCCTAGTGGAGCTAAG