GSM9286572: Day 3 Kupffer cells; Mus musculus; RNA-Seq
Mouse livers were dissociated by perfusion and collagenase digestion and total non-parenchymal cells were isolated by differential centrifugation. Non parenchymal cells were further purified by FACS and live F4/80-high, CD11b-Intermediate cells were used for the single cell RNAseq protocol. The 10X Genomics Single Cell 3' Expression library preparation is performed using the 10X Genomics Chromium Controller. The cells prepared from disassociated tissue or tissue culture are validated for viability and cell concentration using the Countess Automated Cell Counter (Life Technologies) targeting ≥75% cell viability. The cell emulsification is performed on the 10X Chromium Controller using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics PN-120267) and Chromium Single Cell A Chip Kit (10X Genomics PN-1000009). Well 1 of Chip A is loaded with the reverse transcription (RT) Master Mix + cell suspension containing ~16,000 cells to target 10,000 emulsified cells at ~65% efficiency of emulsification. Well 2 of Chip A is filled with 40ul of the GEL Beads. Well 3 of Chip A is filled with 270ul Partitioning Oil. Any unused wells are filled with 50% glycerol at a volume designated for the well number. A gasket is applied to the Chip A and the loading cassette is inserted into the Chromium Controller for GEM creation using the Chromium Chip A Single Cell run program. Emulsified GEMs are recovered from each well and transferred to 200ul strip tubes. The RT reaction to generate 10X barcoded single stranded cDNA, in the single cell containing GEMs, is performed using an Eppendorf MasterCycler Pro thermal cycler. Post GEM-RT Cleanup is conducted by breaking the GEMS with 10X Recovery Agent to separate the aqueous phase from the Recovery Agent and Partitioning oil. A cleanup of the sscDNA containing aqueous phase is completed using Dynabeads MyOne Silane beads (Life Technologies 37002D). The second strand cDNA amplification is performed using the cDNA Amplification Mix on the Eppendorf MasterCycler Pro. The 3' gene expression library construction is initiated with fragmentation, end repair and A-tailing of the dscDNA followed by adapter ligation and a sample index PCR using the Illumina compatible indexed adapters in the Chromium i7 Multiplex kit (10X Genomics 120262). Validation of the single cell library is conducted using the Agilent Tapestation 4200 ScreenTape assay (Agilent 5067-5576). Single cell library quantification is completed using a Roche LightCycle96 using FastStart Essential Green Master (Roche 06402712001) and KAPA Library Quant (Illumina) DNA Standards (KAPA KK4903). Library concentrations are adjusted and pooled to 1.5nm for a final clustering concentration of 300pM on a NovaSeq6000. The sequencing was performed using a NovaSeq6000 100 cycle Reagent Kit (Illumina 20012865) with an asymmetrical sequencing profile ( read 1 - 26 cycle :  i7 index read - 8 cycle : i5 index read – 0 cycle : read 2 – 91 cycles ). Bcl2fastq conversion and demultiplexing is performed using the 10X Genomics Cell Ranger and Loupe Browser software suite.