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: Neonatal sample collection:  1.The neonatal mice were sacrificed by cervical dislocation. 2.Inject 200 μl ice cold PBS (with 2% FCS) using a 1 ml syringe with 26g needle into the abdominal cavity of the neonatal mice. 3.Then massage the peritoneum gently and insert a 26g needle attached to the 1 ml syringe into the peritoneum to collect the fluid. 4.Inject 200 μl ice cold PBS (with 2% FCS) using a 1 ml syringe with 26g needle into the abdominal cavity of the neonatal mice. Then make an incision in the skin of the abdomen, and collect the fluid with pipette. 5.Then adding 200μl DPBS with 2% FBS to the peritoneal cavity and aspirating the fluid. Repeating this steps for three times to collect more peritoneal cells. 6.Spin the collected cell suspension at 400 x g for 8 minutes, 7.Red blood cells were removed using the ACK Lysing Buffer 8.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. 9.Then the CD19 MicroBeads were added to the suspension, and the mixture was incubated at 4℃ for 10 minutes. 10.Applying the cell suspension onto the MS column placed in the magnetic field of a MACS Separator. And the labeled CD19 positive cells were retained on the column. 11.Collecting the positive selected cells for further research by removing the column from the magnetic field and flushing the column with MACS buffer.  12.The collected CD19 positive cells were filtered with 30-μm Cell Strainer and then were counted with the cell auto counter. 13.Centrifuging the cells and resuspending the cells with suitable volume buffer.  (5')GEM generation and reverse transcription1. GEMs are generated by combining barcoded Single Cell 5' Gel Beads, a Master Mix with cells, and Partitioning Oil on a microfluidic chip. To achieve single cell resolution, the cells are delivered at a limiting dilution, such that the majority (~90 – 99%) of generated GEMs contains no cell, while the remainder largely contain a single cell. Immediately following generation of a GEM, the Single Cell 5' Gel Bead is dissolved and any co-partitioned cell is lysed.2. Upon dissolution of the Single Cell 5' Gel Bead in a GEM, oligonucleotides containing (i) an Illumina R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a 13 nt Switch Oligo are released and mixed with cell lysate and a Master Mix that contains reverse transcription (RT) reagents and poly(dT) primers. Incubation of the GEMs then produces barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, the GEMs are broken and the pooled post GEM-RT reaction mixtures are recovered.3. The pooled post GEM-RT reaction mixture contains barcoded first-strand cDNA from poly-adenylated mRNA, as well as leftover biochemical reagents and primers. Silane magnetic beads are used to purify the cDNA from this mixture. Barcoded, full-length V(D)J segments can then be directly enriched from the purified post GEM-RT product by TCR/Ig constant region-specific PCR amplification prior to library construction. Alternatively, the entire purified post GEM-RT product can be amplified by PCR and aliquoted prior to enriching for barcoded, full-length V(D)J segments. (BCR-seq)BCR region enriched library construction1. Perform the cDNA amplification, and clean up the products.2. Amplified cDNA underwent two rounds of target enrichment using nested primer pairs specific for mouse BCR constant regions.3. Clean up the BCR target enrichment 2 from amplified cDNA reaction with the SPRIselect Reagent.4. Quality control were performed on the Alilent Bioanalyser.Enriched Library construction1. Dispense the appropriate volume for 50 ng (Enrichment from cDNA) into each well of a PCR 8-tube strip on a chilled metal block resting on ice.2. Prepare a thermal cycler with the following incubation protocol and initiate the 4°C precool block step prior to assembling the Fragmentation Mix. The incubation protocol is: 4 °C holding, 32 °C for 2 min, 65 °C for 30 min, 4 °C holding.3. Vortex the Fragmentation Buffer. Prepare the Fragmentation Mix on ice. Add the reagents in the order (Nuclease-free water, Fragmentation Buffer, FragmentationEnzyme Blend). Pipette mix thoroughly and centrifuge briefly.4. Dispense 30 µl Fragmentation Mix into each tube containing 20 µl Target Enrichment 2 product. Pipette mix 15 times on ice and centrifuge briefly before returning the tube strip to a chilled metal block resting on ice.5. Transfer the chilled tube strip into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the Fragmentation protocol.Adaptor Ligation6. Prepare Adaptor Ligation Mix by adding the reagents in the order (Nuclease-Free Water, Ligation Buffer, DNA Ligase, Adaptor Mix).7. Remove tube strip containing Fragmentation, End-Repair & A-tailing Product from the thermal cycler.8. Add 50 µl Adaptor Ligation Mix to each tube containing 50 µl sample from the Fragmentation, End Repair and A-tailing Reaction. Pipette mix 15 times and centrifuge briefly.9. Incubate in a thermal cycler at 20 °C for 15 min.Post Ligation Cleanup – SPRIselect10. Vortex the SPRIselect Reagent until fully resuspended. Add 80 μl SPRIselect Reagent (0.8X) to each sample in the tube strip and pipette mix 15 times.11. Incubate the tube strip at room temperature for 5 min.12. Place the tube strip in a 10x™ Magnetic Separator in the High position until the solution is clear. Carefully remove and discard the supernatant.13. Add 200 µl 80% ethanol to the pellet and stand for 30 sec. 14. Carefully remove and discard the ethanol wash. Repeat this step.15. Centrifuge the tube strip briefly and return it to a 10x Magnetic Separator in the low position.16. Remove and discard any remaining ethanol and allow the samples to air dry for 2 min.17. Remove the tube strip from the 10x Magnetic Separator and add 30.5 µl Buffer EB.Pipette mix 15 times.18. Incubate the tube strip at room temperature for 2 min.19. Place the tube strip in a 10x Magnetic Separator in the Low position until the solution is clear.20. Transfer 30 µl of sample to a new tube strip.Sample Index PCR21. Prepare Sample Index PCR Mix by adding the reagents in the order (Nuclease-Free Water, Amplification Master Mix, SI-PCR Primer). Pipette mix thoroughly and centrifuge briefly.22. Add 60 µl Sample Index PCR Mix to each tube containing 30 µl purified Post Ligation sample.23. Add 10 µl of an individual Chromium i7 Sample Index to each well and record theirassignment. Pipette mix 5 times (pipette set to 90 μl) and centrifuge briefly.24. Index the library DNA in a thermal cycler with the following protocol. The protocol is: 98 °C for 45 s, 9 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.25. Store the tube strip at 4°C for up to 72 h or proceed directly to Post Sample Index PCR Cleanup.26. Clean up the sample index PCR product with SPRIselect Reagent.27. Perform the quality control of the cDNA library.