title: GSM7147876: CITE-seq-8wks-ADT; Mus musculus; OTHER
library_construction_protocol: Total bone marrow was obtained by crushing femur, tibia, humerus and pelvis bones. Cells were lineage depleted using Streptavidin Microbead Kit (Miltenti) using two different cocktails of biotinylated antibodies according to each time point. For the 4-weeks timepoint we used Gr-1, CD19, TCRb, Ter119, CD11b and NK1.1 and for the 8-weeks timepoint we used Gr-1, CD19, TCRb and Ter119. After lineage depletion, cells were stained with Zombie Aqua (Biolegend), followed by incubation with 25 different TotalSeq-A antibodies (Biolegend) listed in Supplementary Table 2. Lin-ckit+Tom+ cells (4-weeks time point) and Lin-Flt3+Tom+ (8-weeks time point) were sorted using flow cytometry. From each mice, 6000 cells were collected and loaded into a Chromium Controller (10X Genomics). Library was prepared using Chromium Next GEM Single Cell 3' Reagens Kits v3.1 with Feature Barcoding technology for surface protein protocol (Revision D, (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR and with specific primers amplifying the antibody-derived tags, following Biolegend protocol for TotalSeq-A antibodies. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the antibody-derived tagged cDNAs (180bp). For the mRNA derived cDNA library preparation, we further proceeded according to the manufacturer's instructions. For antibody-derived tagged library, we used the KAPA HiFI HotStart Library Amplification Kit (Roche) with the following primers and amplification program: -ADT-seq library: amplification was performed at 98°C for 2' followed by 16 cycles at 98°C for or 20''; 60°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with Small RNA RPI3 and RPI4 primer as i7 primer (Illumina) for 4- and 8-weeks time points, respectevely. -Hashtag-seq library: amplification was performed at 98°C for 2' followed by 16 cycles 98°C for 20''; 64°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with TruSeq D703_long and D704_long primer as i7 primer (Illumina) for 4- and 8-weeks time points, respectevely. The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC*T*C-3' SI-PCR primer as i5 primer (Illumina) was used for both ADT- and Hashtag- seq library amplification. Following the final bead purification, all three libraries were pooled as 80% mRNA library, 10% ADT library and 10% hashtag library before sequencing. scRNA-seq coupled to CITE-seq