Methocult express

Immunoselected CD34⁺ cells were cultured for 3 days in StemSpan SFEM with CD34 expansion supplement (StemCell Technologies), then plated in Methocult Express (StemCell Technologies) at a density of 4–500 cells per well in a 6-well plate. After ∼14 days, colonies were identified and counted under a microscope. Individual erythroid (BFU-E) colonies were recovered for RNA-Seq.
HPLC and RNA-Seq HPLC and RNA-Seq were performed (DeWitt et al., 2016 (link); Urbinati et al., 2017 (link)); cDNA was synthesized following the Smart-seq2 method (Picelli et al., 2013 (link)) and fragmented with a Covaris apparatus. Indexed sequencing libraries constructed with the ThruPlex DNA-seq kit (Rubicon Genomics) were sequenced on an Illumina HiSeq 4000 for 50 cycles. RNA-seq reads were quantified using kallisto 0.43.1 (Bray et al., 2016 (link)).

To generate CFU colonies from hCD34⁺ cells 72 hours after nucleofection, a fraction of nucleofected cells was plated on methylcellulose medium (Methocult Express [StemCell Technologies, 04437])-covered 96 well plates using cell density that yields <1 colony per well. For analysis of cells after long-term in vivo xenograft, nucleofected cells were xenotransplanted in NOD.Cg-Kit^W-41J Tyr⁺ Prkdc^scid Il2rg^tm1Wjl/ThomJ (NBSGW) mice³⁹ (link) (Jackson Laboratory, 026622) via tail vein injection 72 hours after nucleofection. Eight to 16 weeks after transplantation, hCD34⁺ cells were sorted from the BM by fluorescence-activated cell sorting after staining with anti-human CD34 antibody (BD Biosciences, 348791) and anti-mouse CD45 antibody (BD Biosciences, 553081). A fraction of the sorted hCD34⁺ cells were collected for NGS, and the rest was plated on a methylcellulose medium. The resulting colonies were collected after 3 weeks in culture, genotyped, and used for single telomere length analysis (STELA).⁴⁰ (link) Mouse xenotransplantations were performed under the protocol AUP-2016-12-9389-1, approved by the office of laboratory animal care at the University of California, Berkeley. Donor and replicate information of all HSPC experiments is listed in Table 2.