Repli g ultrafast mini kit

Lysed single cells: Whole genome amplification was performed on single cells using MDA-2 (REPLI-g Single Cell Kit) (150345, Qiagen).¹² (link) A reaction of a total 50 ul volume was incubated at 30°C for 3 h and inactivate the DNA polymerase at 65°C for 3 min.
Lysed subpopulation cells: Whole genome amplification for lysed subpopulation cells was named MDA-1 (REPLI-g UltraFast Mini kit) (150035, Qiagen).¹² (link) A reaction of a total 20 ul volume was incubated at 30°C for 1.5h and inactivate the DNA polymerase at 65°C for 3 min.
Amplified DNAs were directly used or stored at -20°C. The Qubit 3.0 (Life Tech.) was used to measure the concentration of MDA-1 and MDA-2 products. The quality and genome-coverage for PCR were checked by five housekeeping genes located on different chromosomes. Only the products successfully amplified by at least four genes were chosen for exome capture. PCR primers used were as follows: 2p (Forward [F] primer-5’-GTCTTTAGCTGCTGAGGAAATG-3’, Reverse [R] primer-5’-AGCAGAATTCTGCACATGACG-3’), 3p (F-5’-ATTTATTGCAAACTCCCTAATATCA-3’, R-5’-CCTCCATTGGCATGAAGTCT-3’), 4p (F-5’-AACTGAATGGCAGTGAAAACA-3’, R-5’-CCCTAGCCTGTCATTGCTG-3’), 5p (F-5’-GGGTAAGATCCAGAGCCACA-3’, R-5’-CCTCATTCCTTCTCGAAGCA-3’), β-actin (F-5’-GCCAACTTGTCCTTACCCAGAG-3’, R-5’-GCCAGGAACTCCCCAATAAGC-3’).

We amplified the isolated single cells using the Single-Cell Multiple Displacement Amplification protocol reported previously (6 (link)). Briefly, for each single cell, we added 1 μl of exo-resistant random primer (Thermo Fisher Scientific) and 3 μl of lysis buffer (400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA) and incubated on ice for 10 min. We neutralized the lysis buffer by adding 3 μl of stop buffer [400 mM HCl and 600 mM tris-HCl (pH 7.5)]. We then added 32 μl of master mix containing 30 μl of multiple displacement amplification reaction buffer and 2 μl of Phi29 polymerase (REPLI-g UltraFast Mini Kit, QIAGEN), incubated for 1.5 hours at 30°C and 3 min at 65°C, and held at 4°C until purification. We purified the amplicons using AMPure XP beads (Beckman Coulter) and quantified DNA concentration with the Qubit High-Sensitivity dsDNA Kit (Thermo Fisher Scientific). Simultaneously, we amplified 1 ng of genomic DNA in 2.5 μl of PBS as positive control and 2.5 μl of PBS without any template as negative control. We performed the locus dropout test as described previously (6 (link)) with primers designed for each species separately (table S2). Three single-cell amplicons per individual per experimental condition that passed the locus dropout test were prepared for whole-genome sequencing.