skn1_dna_069
single cell dna seq of a skin fibroblast cell line
Immediately after the BAGs were collected and washed, a linear extension step using DNA polymerase I (NEB, M0210) was performed at room temperature for 1.5 hours and then 37C for 30 minutes. That was followed by a 3 exonuclease treatment using Exonuclease I (NEB, M0293) to chew up unused single-stranded primers. The DNA in BAGs were cut using NLAIII (NEB, R0125) to generate a 3CATG overhang. The BAGs were then distributed into a 96-well plate. In each well, we added dNTP (Sigma-Aldrich, 11814362001) and well-specific primers with the following structure: 5 adapter 1 - BAG barcode 1 - varietal tag  CATG. The last two bases (T and G) are locked nucleic acid (LNA) to improve annealing to the four base overhang. We then perfrom what we call a ligation-extention reaction, in which ligation and extension occur in the same reaction. To be specific, after the BAGs were first incubated with 0.8 L of 100 M well-specific primers in 1x Quick Ligation Buffer (NEB, M2200) in a total volume of 14 L at 4C for 20 minutes with rotation, 0.75 L of Quick Ligase (NEB, M2200) and 0.75 L of Klenow Fragment (NEB, M0212) in 4.5 L of 1x Quick Ligation buffer were added. The plate was incubated at 4C for another 20 minutes with rotation and then at 10C for 30 minutes to promote ligation. Following that, the plate was then rotated at room temperature for 40 minutes and then at 37C for another 40 minutes to allow linear extension. The reaction was stopped by high EDTA buffer STOP-2517. The beads were then pooled together and washed by STOP-1017.  The 2nd round of BAG barcode was added in the same way. Although many strategies can be used to add the 2nd and even more rounds of BAG barcode based on the common sequence from the previous round18-21, here we used PCR to add 96 different 2nd BAG barcodes in 96-well plate. In each well, there is a universal PCR primer UP1 and a well-specific primer with the structure: 5 UP2 - BAG barcode 2  adapter 1. A proportion of BAGs from 1st pool-and-split experiment were evenly distributed into 96 wells. DNA BAGs were amplified using NEBNext Ultra II Q5 Master Mix (NEB, M0544). RNA BAGs were amplified using KAPA HiFi HotStart ReadyMix (Roche #KK2602). The PCR product was pooled together and purified using AMPure XP magnetic beads (Bechman Coulter, A63881). Single nuclei were deposited into individual wells in a 96-well plate and amplified using GenomePlex WGA4 kit (Sigma-Aldrich, WGA4-50RXN) according to the manufacturers instructions. WGA DNA was sonicated using a Covaris focus acoustics system.  The Covaris E210 300 sonication program generated WGA DNA inserts of the desired length ~300 bp, (range 200-400 bp) for library construction. Customized well-specific barcodes were ligated to the fragments in each well. Multiple libraries were combined into pools ranging from 8-12 libraries to pools of 96 libraries for 76 bp single-read sequencing on single lanes of Illuminas GAIIx and HiSeq flowcells respectively.  The first 30 bases of each read were trimmed to remove any WGA primer sequence.
skn1_dna_069
SUB6325769
skn1_dna_069