alias: GSM4766541
title: GSM4766541: LSK_old [CITE-seq]; Mus musculus; OTHER
library_construction_protocol: HSPCs from young and old C57BL/6 mice were sorted by flow cytometry. Bone marrow (BM) cells were stained simultaneously with antibodies for sorting and CITE-Seq in three steps. BM cells were incubated with an anti-Clca3a1 antibody (DHSB, 10.1.1), then with a lineage mix of biotinylated antibodies together with an anti-syrian hamster PE/Cy7 antibody and finally the cells were stained with streptavidin eF450, anti-cKit-APC and anti-Sca1-FITC. At this last step all Total-seq antibodies were added: anti-PE (Biolegend, 408109) , anti-CD150 (Biolegend, 115945), anti-CD48 (Biolegend, 103447) + isotype controls. After sorting in PBS 0,04% BSA, cells were washed, counted with an automated cell counter (EVE, NanoEnTek) and adjusted to a concentration of 1000 cells/µL. Libraries were constructed using the Chromium Single Cell 3′ Library Construction Kit v3. ADT-derived cDNAs and mRNA-derived cDNAs were separated based on their size using 0.6X AMPure XP Beads (Beckman Coulter). The mRNA-derived cDNAs contained in the bead fraction were further processed following the standard 10X Genomics protocol for cDNA library generation. The ADT-derived cDNAs contained in the supernatant were further purified as follows: 1.4X beads were added to obtain a final AMPure XP beads concentration of 2X and samples were incubated 10 min at RT. Supernatant was discarded and beads were washed with Ethanol 80%, air-dried and resuspended in 50 uL water. The same procedure was repeated once. Beads were washed twice with Ethanol 80% and air-dried. The purified ADTs were subsequently eluted in 45 mL water at RT for 5 min. To generate the ADT sequencing library, 45 mL ADTs were used as template in a 100 uL PCR reaction with the NEBNext® High Fidelity Master Mix (NEB), a TruSeq small RNA RPIx (containing i7 index) primer and the 10X Genomics SI-PCR primer (see primer table). The PCR products were incubated with 1.6X AMPure XP beads at RT for 5 min. After washing the beads twice with Ethanol 80%, the ADT library was eluted in 30 mL water. The scRNA-Seq libraries and the ADT libraries were sequenced together on an Illumina NextSeq 500 platform.
identifiers.namespace: GEO
identifiers.id: GSM4766541
attributes.tag: GEO Accession
attributes.value: GSM4766541