Anti cd45 percp cyanine5

Uteri were dissected and minced into small fragments. Then, the minced uteri were placed in HBSS containing 200 U/ml hyaluronidase (Sigma‐Aldrich), 1 mg/ml collagenase type IV (Sigma‐Aldrich) and 0.2 mg/ml DNase (Sigma‐Aldrich) and incubated at 37°C for 30 min. as previously described with minor modifications 10. After digestion, the cells were centrifuged and washed with PBS containing 0.2% BSA and incubated in the same buffer for 15 min. at 37°C before filtration through 37 μm nylon mesh. After centrifugation, the cells were resuspended in PBS containing 0.2% BSA for further staining. The cell suspensions were blocked with antimouse CD16/CD32 and then incubated with fluorescently labelled antibody at 4°C for 30 min. The following antibodies were used for flow cytometry analysis: anti‐CD45 PerCP‐Cyanine 5.5 (45‐0451; eBioscience, San Diego, CA, USA), anti‐CD3 PE (12‐0031; eBioscience), anti‐CD49b FITC (11‐0491; eBioscience). After staining, the cells were washed and suspended in PBS containing 0.2% BSA for analysis on a FACScalibur (BD Biosciences, Franklin Lakes, NJ, USA) instrument. The data were analysed using FCS Express V3 software (De Novo Software, Glendale, CA, USA).

For newly sequenced samples with low tumor purity (below 60%), the leukemic cell population was enriched either by flow cytometric sorting or T cell depletion by magnetic beads (EasySep Human CD3 Positive Selection Kit II, 17851, StemCell Technologies). For flow cytometric sorting, CD45^dimCD33^dim positive population was sorted using anti-CD45 PerCP-Cyanine5.5 (eBioscience cat# 8045–9459-120) and anti-CD33 APC (eBioscience cat# 17–0338-42). CD34 gating using anti-CD34 PE (Beckman cat# IM1459U) was added depending on the positivity of each patient sample. Enrichment of the tumor population was confirmed flow cytometric analysis of the post-sorting samples (generally > 90%). Libraries were constructed using the TruSeq Stranded Total RNA Kit, with Ribozero Gold (20020598, Illumina) for RNA-Seq, the TruSeq DNA PCR-Free Library Prep Kit (20015963, Illumina) for WGS, and the TruSeq Exome Kit v1 (20020614, Illumina) for WES according to the manufacturer’s instructions. After library quality and quantity assessment, samples were sequenced on HiSeq2000 or 2500 (Illumina, RRID:SCR_020132, RRID:SCR_016383) instruments with paired-end (2 × 101 bp, 2 × 126 bp, or 2 × 151 bp) sequencing using TruSeq SBS Kit v3-HS (FC-401–3001, Illumina) or TruSeq Rapid SBS Kit (FC-402–4023, Illumina).