Phusion pcr master mix

The ATAC-sequencing libraries were prepared as in (Buenrostro et al., 2015 ). For preparation of the ATAC-seq libraries, the cells were detached by a 5-min TrypLE incubation, washed in cold 5% FBS-PBS, and separated in flow cytometry based on EmGFP expression, which indicates DUX4 expression. 5×10⁴ EmGFP (−) and EmGFP (+) DUX4TetOn-hESCs (H1 clone 2, H1 clone 8, H9 clone 3 and H9 clone 4) were centrifuged at 500×g for 5 min. The pellets were washed in cold 1× PBS by centrifugation at 500×g for 5min. Each cell pellet was lysed in 50 μL of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, and 0.1% IGEPAL CA-630) and centrifuged at 500×g at 4°C for 10 min. The pellet was then resuspended in the transposase reaction mix (2.5 μL of transposase in TD buffer (Nextera DNA library preparation kit, Illumina) and incubated at 37°C for 30 min. The reactions were purified through columns and eluted in 20 μL. After addition of the barcode oligos the DNA samples were amplified for 12 cycles (98°C for 10 s, 63°C for 30 s and 72°C for 60 s) in Phusion PCR master mix (Thermo Fisher Scientific). The PCR products were purified through the columns and eluted in 20 μL. All ATAC-seq libraries were sequenced in single-end mode on an Illumina NextSeq 550 platform using the 75 cycles High Output Kit (v2.5).

Genomic DNA was extracted from sorted hematopoietic cells and amplified using Phusion PCR Master Mix (Thermo Scientific, Waltham, MA). The polymerase chain reaction (PCR) was stopped at the exponential phase, and the PCR product was purified using AMPure XP beads (Beckman Coulter A63880) before high-throughput sequencing. The sequencing data were analyzed as previously described^{29 (link),33 (link),34 (link)}. Barcode data were analyzed using our customized Python scripts^{32 –34 (link)}. We filtered out clones with more than 0.5% white blood cell (WBC) abundance in one cell type at one-time point and absent in all other cell types and time points. Sequencing data were combined with FACS data to calculate the clonal abundance as follows:
Clonal abundance = 100% × (each cell population (Gr, B, CD4 T, or CD8 T cells)% WBCs) × (donor% each cell population) × (GFP% donor cells) × (number of reads for each barcode)/(total reads of all barcodes).

We performed double‐digest restriction site‐associated DNA sequencing (ddRADseq; Peterson et al., 2012 (link)), using the restriction enzymes EcoRI and PstI, both of which use six‐base‐pair recognition sites. We multiplexed individuals with 30 unique adapter barcodes. We size‐selected 500–600 bp fragments using a Pippin Prep Blue (Sage Science) and amplified final libraries using Phusion PCR master mix (Thermo Fisher Scientific). Due to the large genome size of A. barbouri (approximately 24 Gb; www.genomesize.com), we included only 30 individuals in each of four final libraries to attempt to increase sequencing depth across individuals; two individuals from each sampling location were duplicately sequenced in different libraries. A total of four libraries were sequenced on an Illumina HiSeq 2000 sequencing system at the University of Oregon Genomics Core Facility (gc3f.uoregon.edu) using single‐end, 100 bp reads.

We selected a subset of 12 residues present on the predicted chitin-binding surface of ChBD_CHIT1-49 for substitution into alanine. The 12 single mutants of the protein BP-ChBD_CHIT1-49 were generated using the Quick-Change Multi Site-Directed Mutagenesis kit (Agilent Technologies) following the manufacturer’s instructions. Briefly, mixtures of 50% Phusion PCR Master Mix (Thermo Fisher Scientific), 5% DMSO, 100 ng of template DNA and 2 mM of primers were submitted to the following PCR program: 30 s at 98 °C, 10 s at 98 °C, 30 s at 42 °C and 15 s at 72 °C (30 cycles), followed by a final step at 72 °C for 10 min. The PCR products were then digested by DpnI in FastDigest Buffer (Thermo Fisher Scientific) for 10 min at 37 °C to remove template DNA. Finally 10 μL of the resulting mutated plasmids were used to transform E. coli DH5α competent cells for plasmid amplification.

2% HFE oil is composed of HFE (Novec 7500, 3 M) supplemented with 2%(w/w) 008-FluoroSurfactant (RAN Biotechnologies). 5% FC40 oil is FC40 (Sigma) supplemented with 5%(w/w) 008-FluoroSurfactant. 2% HFE oil was used to make a water-in-oil emulsion containing Phusion PCR master mix (M0530S, Thermo Fisher) 0.5 μM of each primer, and 70 fM template DNA (mCherry gene from plasmid pfm301) in the detergent-free buffer (F520L, Thermo Fisher) supplemented with 2.5%(v/v) Tween 20 and 2.5%(v/v) PEG 6000. Flow rates of aqueous and oil phases used for generating droplets were 125 and 250 μl/h, respectively. The oil phase of the collected droplets was exchanged with 5% FC40 oil by instructing the liquid-handling robot to remove oil from the bottom of PCR tubes because the aqueous droplets float on top of the denser fluorinated oil. 5% FC40 oil supports better droplet stability over the course of the PCR thermal cycle. After PCR, the oil phase was exchanged again with 2% HFE oil supplemented with SYBR Green dye (S7563, Thermo Fisher) 1× concentrate to stain the PCR products. The droplets were visualized in a Countess slide (C10228, Invitrogen), which produces a monolayer that is easily imaged on a microscope (EVOS FL, Thermo Fisher) using both transmission and GFP channels. The acquired images were analyzed with the ImageJ software to extract droplet size and fluorescence distributions.

The lentiviral DNA barcode construct and its application have been described in detail previously^{30 (link)}. The DNA barcode was inserted into the 3′UTR region of GFP and transcribed together with GFP. Genomic DNA was extracted from sorted barcoded leukemia cells and amplified using a Phusion PCR master mix (Thermo Scientific, Waltham, MA). The PCR reactions were halted once they had progressed halfway through the exponential phase. PCR product was purified and analyzed using high-throughput sequencing. We combined sequencing data with FACS data to calculate the clonal abundance for each clone as indicated below. Clones with a clonal abundance greater than 0.01% were used for further analyses. $Clonalabundance\%=100*\left[\frac{\#ofreadsforeachbarcode}{totalreadsforallbarcodes}\right]\left[\frac{\#ofhumancells}{totalMNC}\right]\left[\frac{\#ofGFPcells}{totalhumancells}\right]$