title: Sub Library 7
organism: Homo sapiens
description: Protocols: Samples from healthy and diseased teeth were collected from patients attending the Outpatient Dentistry Department and the One-Day Clinic at the Erasme University Hospital (HUB - Erasme site). Immediately after extraction, the samples were placed in Hank's Balanced Salt Solution (HBSS, Sigma-Aldrich, St.Louis, USA) containing 1% Penicillin/Streptomycin/Amphotericin B solution (A5955-100ML, Sigma-Aldrich, St.Louis, USA) and kept on ice. All samples were processed and dissociated within 60 minutes of extraction. Fresh collagenase solution for tissue dissociation was prepared by resuspending 40 mg of Collagenase Type 4 (Worthington Biochemical, NJ, USA) and 4 mg of calcium chloride in 10 mL of prewarmed TrypLE™ Express Enzyme 1X phenol red (Thermo Fisher Scientific, Massachusetts, USA) and maintained at 37°C. Dissection forceps and scissors were disinfected using a VIRKON S solution (Germineo, Onet Le Chateau, FRANCE). Petri dishes were filled with sterile, cold RPMI solution containing 1% Penicillin/Streptomycin/Amphotericin B.  To process the tooth, it was first held using a tooth holder, and the periodontium was scraped off using a surgical blade. The surface of the tooth was carefully wiped with 70% ethanol. The tooth was then cracked using a hammer, ensuring minimal compression to avoid tissue damage. The cracked fragments were placed into a petri dish containing cold RPMI solution and kept on ice.  Under a stereomicroscope and using forceps, the fragments were carefully separated to expose the dental pulp. The pulp was then extracted from the pulp chamber, transferred to a fresh petri dish filled with cold RPMI/antibiotic-antimycotic solution, and kept on ice. The dental pulp tissues were finely chopped into small pieces using fine scissors and immediately resuspended in a sterile 15 mL Falcon tube containing 3 mL of prewarmed collagenase solution.  The tubes were incubated at 37°C for 50 minutes, with manual shaking performed every 10 minutes. After incubation, the tubes were placed on ice, and the tissue was manually triturated by pipetting up and down, first with a slightly cut P1000 tip and then with a normal P1000 tip, for 3 minutes until complete tissue disaggregation.  The cell suspension was filtered through a 40 µm cell strainer into a new 15 mL tube, rinsing the filter with 1 mL of RPMI solution. The tube was centrifuged at 500g for 10 minutes in a swinging bucket rotor. The supernatant was carefully decanted, and the pellet was resuspended in 375 μL of cold Cell Prefixation Buffer (Parse Fixation Kit v2, Parse Biosciences, Seattle, USA).  Next, 125 μL of cold Cell Fixation Solution (ECF2101 Evercode™ Cell Fixation v2, Parse Biosciences, Seattle, USA) was added to the suspension, mixed by pipetting three times with a P1000, and incubated on ice for 10 minutes. Then, 40 μL of cold Cell Permeabilization Solution (ECF2101 Evercode™ Cell Fixation v2, Parse Biosciences, Seattle, USA) was added, mixed, and incubated on ice for 3 minutes. Finally, 500 μL of cold Cell Neutralization Buffer (ECF2101 Evercode™ Cell Fixation v2, Parse Biosciences, Seattle, USA) was added, and the tube was centrifuged at 500 × g for 10 minutes in a swinging bucket rotor.  The supernatant was carefully removed and discarded, and the pellet was resuspended in 75 μL of cold Cell Buffer (ECF2101 Evercode™ Cell Fixation v2, Parse Biosciences, Seattle, USA) and transferred to a 1.5 mL tube. A 10 μL aliquot of the cell suspension was taken for cell counting. This aliquot was mixed with 40 μL of 0.4% trypan blue, and the cells were counted using a Neubauer chamber.  To preserve the sample, 3.75 μL of DMSO (ECF2101 Evercode™ Cell Fixation v2, Parse Biosciences, Seattle, USA) was added, and the sample was stored in a Mr. Frosty freezing container at -80°C for up to 6 months, until further processing for the single-cell protocol.  Single-cell sublibraries were generated following the Evercode™ WT v2 user manual v2.3 (ECW02130 Evercode™ WT v2, Parse Biosciences, Seattle, USA). Samples were thawed, counted, and recorded into the Sample Loading Table provided. Based on the Sample Loading Table values, samples were diluted with Dilution Buffer and loaded into the Round 1 Plate. During this step, an in-situ reverse transcription reaction was performed, and well-specific barcodes were added. The cells were then pooled, centrifuged, and resuspended.  The pooled cells were mixed with the Ligation Master Mix and transferred to the Round 2 Plate, where an in-situ ligation reaction attached a second well-specific barcode to the 3′ end of the cDNA. After pooling and straining, Round 3 Ligation Enzyme was added to the sample, which was then loaded into the Round 3 Plate. During this step, a second in-situ ligation reaction introduced a third well-specific barcode, the Illumina TruSeq R2 sequence, and a biotin to the cDNA. The sample was subsequently pooled, strained, centrifuged, washed, and resuspended in Dilution Buffer.  The cells were counted and allocated into sublibraries following the guidelines in the Sublibrary Generation Table. These sublibraries were lysed and stored at -80°C until further processing. The barcoded cDNA was then captured on streptavidin-coated magnetic Binder Beads and washed to remove cellular debris. After an additional wash, a template switching reaction was performed on the captured cDNA, adding a 5′ adaptor. The captured cDNA was further washed and amplified using primers targeting the template switching (TS) sequence and the Illumina TruSeq R2 sequence.  The amplified cDNA was purified using a 0.8x SPRI bead cleanup. Its concentration and size distribution were assessed using fluorescent dyes (Qubit™ dsDNA HS Assay Kit) and capillary electrophoresis (High Sensitivity DNA Kit on the Agilent Bioanalyzer System). The barcoded and amplified cDNA was subsequently fragmented, end-repaired, and A-tailed in a single reaction. Fragmented and A-tailed DNA underwent size selection with sequential 0.6x and 0.8x SPRI bead cleanups. Adaptors containing the Illumina TruSeq R2 sequence were ligated to the 5′ end of the fragmented DNA, followed by another 0.8× SPRI bead cleanup.  Adaptor-ligated DNA was PCR-amplified using Illumina TruSeq R1 and R2 primers. This indexing PCR created sequencing libraries while adding i5/i7 unique dual indices (UDIs) as a fourth layer of cell barcoding. The sequencing libraries were size selected using a double-sided SPRI bead cleanup. Concentration and size distribution were measured again using fluorescent dyes (Qubit™ dsDNA HS Assay Kit) and capillary electrophoresis (High Sensitivity DNA Kit on the Agilent Bioanalyzer System). Finally, the sublibraries were stored at 4°C for up to 48 hours or at -20°C for up to 3 months. Sublibraries were submitted for sequencing with the NovaSeq X Plus Series (PE150, Novogene UK, Cambridge, UK).