CITE-seq of LSK cells from aged mice after long-term iron chelator regimen

We found that iron chelation restored functional defects in aged HSC, including engraftment potential and platelet bias. To gain molecular insights into iron-dependent mechanism for sustaining HSC identity during aging, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) with lineage (Lin)− Sca-1+ cKit+ (LSK) cells isolated from aged mice after long-term regimens with iron chelator Deferoxamine or vehicle control. To investigate the effect of long-term iron chelation during HSC aging, mice aged ~6 months were subjected to iron chelator (Deferoxamine, 50 mg/kg) or vehicle control (HBSS) daily regimens for consecutive 7 days, repeated every 4 weeks for a total of 13 months. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) was performed to examine the molecular regulation by iron chelation. Briefly, lineage depleted bone marrow cells were stained for 30 min with antibodies for lineage, Sca-1, c-Kit, TotalSeq-C Mouse Universal Cocktail and sample-specific hashing antibodies. Stained cells were then sorted on Moflo Astrios EQ (Beckman Coulter), and approximately 20,000 Lin-Sca1+ckit+ (LSK) cells were acquired per sample. Single cell CITE-seq libraries were generated using the 10x Genomics Chromium Next GEM Single Cell 5’ Reagent Kit v2 (10x Genomics, Pleasanton, CA, USA). FACS-sorted LSK cells from bone marrow were stained and pooled in equal amounts prior to chip loading. Libraries were constructed according to manufacturer instructions. Libraries were purified and paired-end sequenced using NovaSeq 6000 S4 Reagent Kit v1.5 with 150 cycles (Illumina, San Diego, CA, USA).