title: Illumina NovaSeq 6000 paired end sequencing; Single cell RNA-seq and BCR-seq of peritoneal B cells
design: Single cell RNA-seq and BCR-seq of peritoneal B cells
library_construction_protocol: Adult and elderly sample collection: 1. The mice were sacrificed by cervical dislocation, spraying it with 70% ethanol. 2. Use a scissors and forceps to cut the outer skin of the peritoneum and gently pull it back to expose the inner skin lining the peritoneal cavity. 3. Inject 10 ml of ice cold PBS (with 2% FCS) into the peritoneal cavity with a 27g needle. Push the needle slowly in the peritoneum. 4. After injection, gently massage the peritoneum to dislodge any attached cells into the PBS solution. 5. Insert a 25 g needle, bevel up, attached to a 10 ml syringe in the peritoneum and collect the fluid while moving the tip of the needle gently to avoid clogging by the fat tissue or other organs. Collect as much fluid as possible and depositing the collected cell suspension in tubes kept on ice after removing the needle from the syringe. 6. Repeat step 3-5. 7. Make an incision in the inner skin of the peritoneum and use a pipette to aspirate the fluid.  8. Spin the collected cell suspension at 400 x g for 8 minutes, 9. Red blood cells were removed using the ACK Lysing Buffer 10. Centrifuge the lavage fluid of the peritoneal cells at 400 x g for 6 minutes and resuspending the cell pellet in 90 μl MACS Buffer which is prepared by dissolving bovine serum protein with auto MACS Rinsing Solution to form a 0.5% BSA solution. 11. Then the CD19 MicroBeads were added to the suspention, and the mixture was incubated at 4℃ for 10 minutes. 12. Applying the cell suspention onto the MS column placed in the magnetic field of a MACS Separator. And the labeled CD19 positive cells were retained on the column. 13. Collecting the positive selected cells for further research by removing the column from the magnetic field and flushing the column with MACS buffer.  14. The collected CD19 positive cells were filterd with 30-μm Cell Strainer and then were counted with the cell auto counter. 15. Centrifuging the cells and resuspending the cells with suitable volume buffer to ensure that the concentration is 3 x 105 – 1 x 106 cells/ml.  3' v3 Nucleic extraction (3ʹ v3.1)1. GEMs were generated by combining barcoded Single Cell 3ʹ v3.1 Gel Beads, a Master Mix containing cells, and Partitioning Oil onto Chromium Next GEM Chip G. To achieve single cell resolution, cells were delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contained no cell, while the remainder largely contained a single cell. 2. Immediately following GEM generation, the Gel Bead was dissolved, primers were released, and any co-partitioned cell was lysed. Primer containing: an Illumina TruSeq Read 1 (read 1 sequencing primer), 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI), 30 nt poly (dT) sequence. Primers were mixed with the lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA.3. After incubation, GEMs were broken and pooled fractions are recovered. Silane magnetic beads were used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which included leftover biochemical reagents and primers. Barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction. Firstly, Perform the cDNA amplification, and clean up the products. Fragmentation, End Repair & A-tailing 1. Vortex Fragmentation Buffer. Verify there is no precipitate. Prepare Fragmentation Mix (Fragmentation Buffer, Fragmentation Enzyme) on ice. Pipette mix and centrifuge briefly.  2.Transfer only 10 μl purified cDNA sample from cDNA Cleanup to a tube strip. Add 25 μl Buffer EB to each sample. And add 15 μl Fragmentation Mix to each sample. Pipette mix 15x on ice. Centrifuge briefly.  3. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.  Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect 4. Vortex to resuspend SPRIselect reagent. Add 30 μl SPRIselect (0.6X) reagent to each sample. Pipette mix 15x. Incubate 5 min at room temperature.  5. Place on the magnet High until the solution clears.  6. Remove 80 μl supernatant  7. Add 125 μl 80% ethanol to the pellet. Wait 30 sec. Remove the ethanol. Repeat this step.  8. Centrifuge briefly. Place on the magnet•Low until the solution clears. Remove remaining ethanol.  9. Remove from the magnet. Add 50.5 μl Buffer EB to each sample. Pipette mix 15x.  10. Incubate 2 min at room temperature.  11. Place on the magnet•High until the solution clears.  12. Transfer 50 μl sample to a new tube strip.  Adaptor Ligation 13. Prepare Adaptor Ligation Mix (Ligation Buffer, DNA ligase, Adaptor Oligos) 14. Add 50 μl Adaptor Ligation Mix to 50 μl sample. Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly.  15. Incubate at 20 °C for 15 min in a thermal cycler. Post Ligation Cleanup –SPRIselect 16. Vortex to resuspend SPRIselect Reagent. Add 80 μl SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 μl).  17. Incubate 5 min at room temperature. Place on the magnet•High until the solution clears. Remove the supernatant.  18. Add 200 μl 80% ethanol to the pellet. Wait 30 sec. Remove the ethanol. Repeat this step. 19. Centrifuge briefly. Place on the magnet•Low.  20. Remove any remaining ethanol. Air dry for 2 min.  21. Remove from the magnet. Add 30.5 μl Buffer EB. Pipette mix 15x.  22. Incubate 2 min at room temperature.  23. Place on the magnet•Low until the solution clears.  24. Transfer 30 μl sample to a new tube strip.  Sample Index PCR 25. Prepare Sample Index PCR Mix. Add 60 μl Sample Index PCR Mix to 30 μl sample.  26. Add 10 μl of an individual Single Index to each well and record the well ID used. Pipette mix 5x. Centrifuge briefly.  27. Incubate in a thermal cycler with the following protocol. The protocol is : 98 °C for 45 s, several cycles of 98 °C for 20 s, 54 °C for 30 s, and 72 °C for 20s, 72 °C for 1 min, and 4 °C holding. Post Sample Index PCR Double Sided Size Selection – SPRIselect 28. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x. Incubate 5 min at room temperature.  29. Place the magnet•High until the solution clears.  30. Transfer 150 μl supernatant to a new tube strip.  31. Vortex to resuspend the SPRIselect reagent. Add 20 μl SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 μl).  32. Incubate 5 min at room temperature.  33. Place the magnet•High until the solution clears.  34. Remove 165 μl supernatant.  35. With the tube still in the magnet, add 200 μl 80% ethanol to the pellet. Wait 30 sec. Remove the ethanol. Repeat this step.  36. Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol.  m. Remove from the magnet. Add 35.5 μl Buffer EB. Pipette mix 15x.  37. Incubate 2 min at room temperature.  38. Place on the magnet•Low until the solution clears.  39. Transfer 35 μl to a new tube strip.  40. Perform the quality control of the cDNA library.