Flow sorted chromosome libraries, chromosome 5 of Homo sapiens: CHM13 Expt 1
Chromosomes were labeled for 2 hours with 50 g/ml Chromomycin A3 and 5 g/ml Hoechst 33258 in the presence of 25 mM sodium sulfite, 10 mM sodium citrate and 10 mM MgSO4. Before sorting, chromosome suspension was filtered through an 8 m strainer. Chromosomes were sorted on a MoFlo Legacy (Beckman Coulter) at 60-65 psi using a 70 m tip. Hoechst 33258 was excited with a UV laser (330-360nm) and the emission was detected in the 409-480 nm range. Chromomycin A3 was excited with the second laser tuned to 458 nm and emission was detected in a 505-605 nm range. Clumps and debris were excluded by gating on forward scatter versus pulse width (fig. S33). Chromosomes were sorted into DNA LoBind collection tubes (Eppendorf). Approximately 37000-123000 sorted events were collected per chromosome. Data analysis was performed using FlowJo (BD Life Sciences).Genomic DNA was extracted from sorted chromosomes using the MagAttract HMW DNA Kit (Qiagen). DNA was analyzed and quantified using a FemptoPulse (Agilent). Whole-chromosome shotgun libraries were prepared from one nanogram DNA using the Accel-NGS 2S DNA Library Kit (Swift Biosciences) with unique dual index adapters according to manufacturers protocol with the substitution of Kapa HiFi HotStart Ready Mix (Roche) for library amplification. Twelve cycles of PCR were used for samples with 2 ng DNA input and 15 cycles were used for the sample with 0.17 ng input DNA. The libraries were pooled for sequencing on a HiSeq 2500 DNA sequencer in Rapid mode (Illumina, Inc.), generating 336 M paired-end 151 bp reads.