title: GSM9059447: Mouse Lung, STFK subunit vaccine immunization, replication 3; Mus musculus; RNA-Seq
library_name: GSM9059447
library_construction_protocol: Excise the lungs into cold PBS. Mince tissue finely, then digest in a pre-warmed enzymatic cocktail at 37°C for 20-45 min with gentle agitation. Quench digestion with cold FBS. Mechanically dissociate further by pipetting and pass the suspension sequentially through 70µm and 40µm cell strainers. Centrifuge, resuspend in ACK lysis buffer for 1-2 min on ice to lyse red blood cells, then immediately quench and centrifuge. Wash the pellet twice in cold PBS buffer . Resuspend in buffer containing RNase inhibitor. Assess cell count and viability (>85%), adjust concentration for the scRNA-seq platform, and keep on ice for immediate processing. 1. Cell Counting & Quality Control: The prepared cell suspension was counted using TC20 Automated Cell Counter (Bio-Rad, USA) to obtain the information of cell size, concentration and viability, and to determine the sample loading volume. 2. GEM Generation and cDNA Amplification: Within the Chromium instrument, barcoded Single Cell 5' Gel Beads are mixed with cells or nuclei, enzymes, and partitioning oil to form tens of thousands of single cell emulsion droplets called “GEMs” (Gel Bead-in-emulsion). Immediately following GEM generation, the Gel Bead is dissolved and any co-partitioned cell is lysed. Oligonucleotides containing (i) an Illumina Read 1 (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt unique molecular identifier (UMI), and (iv) 13 nt template switch oligo (TSO) are released and mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents and poly(dT) RT primers. Incubation of the GEMs produces 10x Barcoded, full-length cDNA from poly-adenylated mRNA. Then, the first-strand cDNA of 10X Barcoded was purified by silane magnetic beads, and sufficient mass was obtained by PCR amplification. The PCR products were clean up by SPRI beads, and double-stranded cDNA was obtained. For VDJ sequencing A small amount of dsDNA was selected for nest amplification using BCR specific primers. Then quantify and qualify the PCR products by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), so as to confirm the input amount of template and the final PCR cycle number for library construction. 3.Library Construction : 50ng PCR products was then treated with Fragmentation Mix to fragment cDNA, End Prep Enzyme Mix to repair both ends and add a dAtailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Each sample was then amplified by PCR for 9 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform Illumina bridge PCR. And P7 primers are a mixture of four primers, each carrying a eight-base index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).