GSM8252375: CITE_Titration_HSC-MPP HTO; Mus musculus; OTHER
CITE-seq was performed with 3 different ADT panel configurations: (1) a customized 65-plex cocktail, (2) a prototype 200-plex cocktail for antibody titration (cat number), and (3) a titrated 110-plex cocktail (cat number). Each configuration used TotalSeq-A antibodies purchased from BioLegend (Supplmentary Table X). The 65-plex cocktail was comprised of known hematopoietic stem and progenitor markers as an initial test of the technology, using CD117-enriched mouse bone marrow as input and applying antibodies at the concentrations recommended by BioLegend. The 200-plex cocktail was provided as a lyophilized mix, uniform for each TotalSeq antibody, which was rehydrated at 8x concentration and subsequently diluted to five concentrations: 4x, 2x, 1x, 0.5x, and 0.25x. 1x concentration was determined based on the performance of ADTs in the 65-plex panel or according manufacturer's recommendation. Five sorted populations were profiled with the 200-plex panel to comprehensively represent rare cell populations in the marrow: total bone marrow cells, CD117-enriched bone marrow cells, FACS sorted HSC/MPP cells (Lin- CD117+ Sca1+ and exclusive of CD48+ CD150-), FACS sorted Multi-Lin cells (Lin- CD117+ CD34+ CD16/32+/low CD115- CD11b- Ly6C- CD150-), and FACS sorted CD127+ (Lymphoid) cells. An unique HTO antibody was added to denote each titration concentration. Same cell population stained with different titration concentrations were pooled together after washing seperately. For the titrated 110-plex panel, a customized lyophilized cocktail was created by BioLegend using selected concentrations from the 200-plex titration experiment. The titrated cocktail was applied to the same five populations except for total bone marrow cells. In addition, each sorted cell population from various gates were stained with an unique HTO, washed and pooled. In each experiment, cells were washed on Laminar Wash MINI or HT2000 System (Curiox Biosystems Inc) with the following settings: 25 cycles, flow rate of 10uL/s, initial volume of 55uL. Pooled cells were captured by 10X Chromium X 3' version 3.1 kit (1000268, 10X Genomics). TEA-seq was performed on the same groups of cells profiled by CITE-seq following it's published (reference eLIFE TEA-seq paper) protocol with minor modifications: (1) using a digitonin concentration of 0.025% based on optimization results, (2) the addition of protector RNAse inhibitor (Roche 3335402001) in perm, wash and tagmentation buffers. (3) 80ul instead of 160ul supernant from cDNA amplifications were used to purify pre-amplified ADT. Briefly, 100,000 to 250,000 leftover cells from CITE-seq experiment were permeablized in digitonin perm buffer, washed and then processed as nuclei according to 10X Multiome (1000283, 10X Gemonics) protocol. Library prep was performed according to protocols with minor modifications such as reduced PCR cycles for ADT and HTO library amplifications. We found pre-PCR ADT/HTO in our libraries were abundant enough that we generally reduced 2 cycles (ADT: 10-12 cycles, HTO: 9-10 cycles). Final transcriptome, ADT and HTO libraries were quantified and analyzed by Qubit dsDNA HS (1000268, Invitrogen), High Sensitivity DNA kit (5067-4626, Agilent Technologies) on 2100 bioanalyzer (G2939BA, Agilent Technologies) and KAPA HiFi library quantification kit (KK4824, Roche). Dual-indexed transcriptome libraries were pooled and sequenced across multiple Illumina Novaseq 6000 S4 or X plus flow cells (Illumina) with PE150+10+10 or PE100+10+10 settings. Single indexed ADT and HTO libraries were pooled and sequenced on Illumina S2 flow cells with PE50+8 setting or with transcriptome libraries. BCL files were demultiplexed into fastq files for CellRanger input. “AT” was added to the end of RPI-x ADT i7 index (6bp) to match D70X_long HTO index (8bp). HTO and ADT FASTQ files were supplied as 3P feature barcode together with transcriptome FASTQ files into Cell Ranger V6.1.2 count pipeline. Transcriptome was mapped to mm10-v2 reference genomes for downstream analysis and visualization.