title: GSM4005128: H10; Mus musculus; RNA-Seq
library_construction_protocol: Cells were isolated from the heart of mice at 1 (n=5), 3 (n=5) and 5 days post-MI (n=3) as described above. Fc-Block was performed (10µg/ml purified rat anti-mouse CD16/CD32, Biolegend TruStain FcX anti-mouse), after which cells were stained with Fixable Viability Staining e780 (1:1000) and CD11b (M1/70) antibodies: CD11b-BV510 (Day 1 cells, 1:300, BD Biosciences), CD11b-Percp-Cy5.5 (Day 3 cells, 1:300, BD Biosciences) or CD11b-PE (Day 5 cells, 1:300, BD Biosciences) for 25 minutes at 4°C in PBS containing 1% FCS. Cells were washed once, resuspended in 1ml of PBS-1%FCS, pooled and centrifuged once more (8', 350rcf). Viable/CD45.2 (intravenous)-negative cells labeled with CD11b-BV510, CD11b-Percp-Cy5.5 and CD11b-PE were separately sorted using a FACS Aria III (BD Biosciences). After sorting, cells were washed once in 1ml of PBS-1%FCS (8', 350rcf). Cells were then separately labeled with hashtag antibodies (Biolegend TotalSeq-A antibodies, Day 1: Hashtag 1, Day 3: Hashtag 2, Day 5: Hashtag 3) and CITE-Seq antibodies (Biolegend TotalSeq-A against mouse Ly6C, Ly6G, CD64, F4/80, CD11c, MSR1, IA/IE, TIM4, CX3CR1, and CCR2). Labeling was performed for 30 minutes at 4°C in 150µl PBS/1%FCS containing 1.5µg of each antibody. Cells were washed with 2ml PBS/1%FCS once (8', 350rcf), resuspended in PBS supplemented with 0.04% BSA, pooled and centrifuged again (8', 350rcf). The cells were resuspended in 150µl PBS/0.04% BSA, filtered through a 40µm cell strainer and counted before loading in the 10X Genomics Chromium. Cells were loaded in the 10X Genomics Chromium at concentrations recommended by the manufacturer. Libraries were prepared with Chromium Single Cell 3' Reagents Kit v3 Chemistry and prepared for CITE-Seq/Hashing according to the manual until the cDNA amplification step in which 1 μl (0.2 μM) ADT PCR additive primer to capture Antibody-derived tags (ADTs) and 1 μl (0.1 μM) HTO PCR additive primer to capture Hashtag oligos (HTOs) were added. After cDNA amplification 60 μl (0.6x) SPRI beads (Beckman Coulter) were added to separate the supernatant fraction that contains the ADT/HTO-derived cDNAs (<180bp) and the bead fraction that contains the mRNA-derived cDNAs (>300bp). The mRNA-derived cDNAs (bead fraction) were processed following the standard 10x Genomics protocol. The ADT/HTO-derived cDNAs (supernatant fraction) were purified twice with 2x SPRI. After the two purifications half amount of the eluted cDNAs were amplified for ADTs and half for hashtags. In the same reactions, the ADTs were indexed using TruSeq Small RNA primers and the hashtags using modified TruSeq DNA primers. The libraries were purified once more with 1.6x SPRI (160 μl). The detailed protocol including the oligo sequences can be accessed here: https://cite-seq.com/protocol
attributes.tag: GEO Accession
attributes.value: GSM4005128