For the isolation of single HSC, BM cells were stained as described
above. During FACS sorting, individual cells were deposited into 8-tube PCR
stripes containing in each well 25 μl lysis buffer (12.6 μg
proteinase K (Thermo Fisher Scientific) in PCR buffer 1 (Roche)). Lysis was done
for 1 h at 55 °C, terminated at 95 °C for 10 min, and cooled to 4
°C before adding the remaining PCR reagents to a final volume of 50
μl. The Polylox cassette was then amplified by nested
PCR. First round PCR: primer #2450 (see above) and primer #494
(5′-AGCTACAGCCTCGATTTGTGGTG-3′) for 5 min at 95 °C; (30 s
at 95 °C, 30 s at 56 °C, 5 min at 72 °C) 35 times; 10 min
at 72 °C. Second round PCR: 1-2 μl of first PCR reaction was used
as template and amplified with primers #2426
5′-CGACGACACTGCCAAAGATTTC-3′ and #2427 (see above) for 5 min at 95
°C; (30 s at 95 °C, 30 s at 62 °C, 5 min at 72 °C)
35 times; 10 min at 72 °C. The nested PCR products were purified by
QIAquick PCR Purification Kit (Qiagen), analyzed by gel electrophoresis for
product length, and analyzed by Sanger sequencing (GATC Biotech). Barcodes were
decoded for each of these sequences.
above. During FACS sorting, individual cells were deposited into 8-tube PCR
stripes containing in each well 25 μl lysis buffer (12.6 μg
proteinase K (Thermo Fisher Scientific) in PCR buffer 1 (Roche)). Lysis was done
for 1 h at 55 °C, terminated at 95 °C for 10 min, and cooled to 4
°C before adding the remaining PCR reagents to a final volume of 50
μl. The Polylox cassette was then amplified by nested
PCR. First round PCR: primer #2450 (see above) and primer #494
(5′-AGCTACAGCCTCGATTTGTGGTG-3′) for 5 min at 95 °C; (30 s
at 95 °C, 30 s at 56 °C, 5 min at 72 °C) 35 times; 10 min
at 72 °C. Second round PCR: 1-2 μl of first PCR reaction was used
as template and amplified with primers #2426
5′-CGACGACACTGCCAAAGATTTC-3′ and #2427 (see above) for 5 min at 95
°C; (30 s at 95 °C, 30 s at 62 °C, 5 min at 72 °C)
35 times; 10 min at 72 °C. The nested PCR products were purified by
QIAquick PCR Purification Kit (Qiagen), analyzed by gel electrophoresis for
product length, and analyzed by Sanger sequencing (GATC Biotech). Barcodes were
decoded for each of these sequences.