Single cell DNA-seq of Homo sapiens; colorectal organoids, APKS, passage 18, replicate 2, plate 1-1
Cells were sorted into 96-well plate with 5 ul of mineral oil (Sigma-Aldrich). After sorting cells can be stored at -20C. 500 nl of lysis mix (0.0005 u Qiagen Protease in NEB Buffer 4) was added to each well and lysis was performed at 55C overnight followed by heat inactivation for 20 minutes at 75C and for 5 minutes at 80C. 500nl of Restriction Enzyme mix (0.5 u NLAIII in NEB Cutsmart buffer) was added to each well and restriction was performed for 3 hours at 37C followed by heat inactivation for 20 minutes at 65C. 100 nl of 1 uM barcoded double stranded NLAIII adapter was added to each well. 1100 ul of Ligation mix (200 u T4 DNA Ligase in 1x T4 DNA Ligase buffer supplemented with 3 mM ATP) was added to each well and ligation was performed overnight at 16C. After ligation single cell were pooled and library preparation was performed as described in Muraro et al. (Muraro et al., 2016). Libraries were sequenced on an Illumina Nextseq500 with 2 x 75 bp paired end sequencing or on a HiSeq 10X with 2 x 150 bp paired end sequencing. Barcoded using 3bp UMI and 96 single cell barcodes (https://github.com/BuysDB/SingleCellMultiOmics/blob/master/singlecellmultiomics/modularDemultiplexer/barcodes/lennart96NLA.bc). 4 libraries per plate.