title: GSM8196012: HUVEC+DPSC+CM  fibrin tissue day 10 sample1; Homo sapiens; RNA-Seq
library_construction_protocol: 10x sample preparation of macrophages suspension: 1 mL of cold lysis buffer comprising 0.32M Sucrose, 5mM CaCl2, 3mM Mg(Ac)2, 20mM Tris-HCl pH 7.5, 0.10% Triton X-100, 0.1mM EDTA pH 8.0, DEPC Water, 40U/mL RNase Inhibitor (Millipore Sigma, cat. No. 3335402001) was added to the frozen cell pellet for 5-minute lysis. The pellet was dissociated by pipette mixing. After the 5-minute lysis, nuclei were pelleted by spinning at 4C for 10 minutes at 800 x g. The supernatant was removed, and the nuclei pellet was resuspended with 2 mL wash buffer comprising 1x PBS, 1% BSA, and 0.1U/uL RNase Inhibitor. Centrifugation and resuspension with wash buffer was repeated for a total of 2 washes. After the second wash, the nuclei suspension was filtered using a 40um Flowmi cell strainer (Millipore Sigma, cat. no. BAH136800040). The purified nuclei suspension was centrifuged at 4C for 10 min at 800 rcf and the nuclei pellet was resuspended in wash buffer for a final nuclei concentration of 1000 nuclei/uL. Nuclei counting was done using AOPI (Revvity, cat. no. CS2-0106-25ML) on a haemocytometer (Thermo Fisher, cat. no. 22-600-100). Following counting, the appropriate volume for each sample was calculated for a target capture of 10,000 cells and loaded onto 10x single cell G chip.  After droplet generation, samples were transferred to a pre-chilled strip tube (USA Scientific, US14024700), and incubated in a Bio-Rad thermos cycler. Sample cDNA was recovered using Recovery Agent provided by 10x and subsequently cleaned up using a Silane DynaBead (Thermo Fisher) mix as outlined by the user guide 3' Reagent Kits v3.1 . Purified cDNA was amplified for 11 cycles before being cleaned up using SPRIselect beads (Beckman Coulter). Samples were run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. 10x sample preparation of tissue samples:  Nuclei were isolated using 10x Genomics' Chromium Nuclei Isolation Kit (10x Genomics, cat. no. 1000493). The frozen tissue was transferred to a 2mL tissue grinder (Millipore Sigma, cat. no D8938). 500ul of Lysis buffer comprised of Lysis Reagent (10x Genomics, cat. no. 2000558), Reducing Agent B (10x Genomics, cat. no. 2000087), and Surfactant A (10x Genomics, cat. no. 2000559) was added to the frozen tissue on ice. The tissue was homogenized using 5 strokes with the loose pestle (pestle A) and 5 strokes with the tight pestle (pestle B). The sample was incubated on ice for a 10-minute lysis. At the end of the lysis incubation, the lysate was transferred to the Nuclei Isolation Column (10x Genomics, cat. no. 2000562) and spun at 16,000 rcf for 20 seconds at 4C. The supernatant was removed and the nuclei pellet was resuspended in Debris Removal Buffer comprised of Debris Removal Reagent (10x Genomics, cat. no. 2000560) and Reducing Agent B. The sample was spun at 700 rcf for 10 minutes at 4C. The supernatant was removed the purified nuclei pellet was washed twice with Wash Buffer comprised of 1x PBS, 10% BSA and RNase Inhibitor (Millipore Sigma, cat. No. 3335402001). The purified nuclei suspension was centrifuged at 4C for 5 min at 500 rcf and the nuclei pellet was resuspended in wash buffer for a final nuclei concentration of 1000 nuclei/uL. Nuclei counting was done using AOPI (Revvity, cat. no. CS2-0106-25ML) on a haemocytometer (Thermo Fisher, cat. no. 22-600-100). Following counting, the appropriate volume for each sample was calculated for a target capture of 10,000 cells and loaded onto 10x single cell G chip.  After droplet generation, samples were transferred to a pre-chilled strip tube (USA Scientific, US14024700), and incubated in a Bio-Rad thermos cycler. Sample cDNA was recovered using Recovery Agent provided by 10x and subsequently cleaned up using a Silane DynaBead (Thermo Fisher) mix as outlined by the user guide 3' Reagent Kits v3.1 . Purified cDNA was amplified for 11 cycles before being cleaned up using SPRIselect beads (Beckman Coulter). Samples were run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3' Reagent Kits v3.1 user guide with modifications to the PCR cycles based on the calculated cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3' Reagent Kits v3.1 user guide with modifications to the PCR cycles based on the calculated cDNA concentration. Sequencing – The molarity of each library was calculated based on library size as measured bioanalyzer (Agilent Technologies) and qPCR amplification data (Roche, cat. no. 07960140001). Gene Expression libraries were sequenced on Illumina's NovaSeqX with the following run parameters: read 1 – 28 cycles, read 2 – 90 cycles, index 1 – 10 cycles and index 2 – 10 cycles.