title: Illumina MiSeq paired end sequencing; Single cell genotyping of Myeloid Leukaemia of Down Syndrome cells
design: Single cell genotyping of Myeloid Leukaemia of Down Syndrome cells
library_construction_protocol: Neonates with Transient Abnormal myelopoiesis (TAM) were (i) sourced within 3 months from birth (majority under 14 days of age), (ii) positive for a GATA1 variant by deep sequencing/conventional Sanger sequencing and (iii) evidence of subsequent clinical and/or GATA1 mutation resolution. ML-DS samples were allocated if ≥3 months from birth, positive for GATA1 variant (by criteria above), and ML-DS relapse if post treatment and GATA1 mutation positive. For sequential samples, “Progressive TAM” refers to samples taken 3 months from birth where neonates still had a clinical diagnosis of TAM, irrespective of treatment status.  Single human or mouse cells were lysed and whole genome amplified using a REPLI-g Single Cell Kit (Qiagen).  Primers for targeted resequencing of patient mutations were designed to include patient-specific heterozygous SNPs within the same amplicon (listed in Table X). For the murine leukemia the primers from the CRISPR-Cas9 screening were used (listed in table X).Targeted amplification was performed using KAPA2G Fast Multiplex PCR Kit (Sigma-Aldrich), followed by addition of sample barcodes and sequencing adapters using FastStart High Fidelity PCR System (Roche) and Access Array™ Barcode Library for Illumina® Sequencers (Fluidigm), as described previously (1).