A protocol for simultaneous high-sensitivity genotyping and chromatin accessibility profiling in single cells

Abstract

Single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) resolves the heterogeneity of epigenetic states across cells but does not typically capture exonic mutations, which limits our knowledge of how somatic mutations alter chromatin landscapes. Here, we present a plate-based approach coupling high-sensitivity genotyping of genomic loci with high-content scATAC-seq libraries from the same single cells. We first describe steps for optimization of genotyping primers, followed by detailed guidance on the preparation of both scATAC-seq and single-cell genotyping libraries, fully automated on high-throughput liquid handling platforms. For complete details on the use and execution of this protocol, please refer to Turkalj, Jakobsen et al.¹.

Keywords: Bioinformatics; Cancer; Genetics; Genomics; Molecular Biology; Sequence Analysis; Sequencing; Single Cell; Stem Cells.

Graphical abstract

Figure 1

Optimal PCR cycle number titration (A) Procedures for establishing the optimal number of PCR cycles for a GTAC experiment. Blue numbers indicate the order in which the steps are executed. (B) SYBR Green-based qPCR was performed after 8 initial cycles of amplification. Each curve represents a single well. The optimal additional number of PCR cycles is highlighted in green.

Figure 2

Bioanalyzer traces showing optimal scATAC-seq libraries For each trace, a variable number of single-cell libraries was pooled and purified. Successful libraries display the characteristic ‘wavy’ shape, with periodic rises and drops in signal intensity (nucleosomal pattern).

Figure 4

Representative gel of primer testing with bulk gDNA Left gel: each lane shows the PCR product of a single primer pair used on gDNA. Arrows are color-coded based on the quality of the product. An optimal primer pair displays amplification as indicated by the green arrow. Right gel: a primer pair for which there is prior knowledge of good amplification is used as positive control. A no-template reaction is used as negative control. A 100 bp ladder was used for both gels.

Figure 3

Schematic overview of primers to be tested prior to the GTAC experiment

Figure 5

Schematic overview of primer testing in single cells ATAC traces are tested from pools of 24 single cells. Efficiency of single-cell genotyping is tested individually in single cells. Blue numbers indicate the order in which the steps are executed.

Figure 6

Single-cell genotyping testing results Primers for each locus were tested in 6 single cells. Each lane represents amplification from a single cell. Depending on the quality and consistency of amplification, primers can be used in a GTAC experiment, or new primers need to be tested for the locus. Bulk gDNA was used as positive control. A no-template reaction was used as negative control.

Figure 7

The automated preparation of barcoded lysis buffer Liquid handling platforms used at individual steps, as well as volumes transferred, are indicated next to the arrows. Blue numbers indicate the order in which the steps are executed.

Figure 8

MANTIS operating instructions (A) Pipetting liquid into a non-filtered tip inserted into the HV chip. (B) Steps for setting up manual priming with water. Numbers indicate the order in which steps are executed. (C) Dispense list display. Numbers in brackets indicate the order in which steps are executed. (D) Chip properties display. Red circles indicate the parameters that need to be defined by the user. (E) Icons used for priming, washing, and volume recovery from the chip.

Figure 9

INTEGRA VIAFLO components Top: components of the VIAFLO. Bottom left: components of the VIAFLO controller. Bottom right: setup for volume transfer between plates. Note that this instrument has 3 plate holders, but an instrument with 2 plate holders can also be used.

Figure 10

Sony MA900 cell sorter calibration (A–D) Panels A and B provide the visual guidelines for calibrating the sorter for sorting into microcentrifuge tubes. Panels C and D provide the visual guidelines for calibrating the sorter for sorting into 384-well plates.

Figure 11

FACS gating strategy for single-nucleus sorting

Figure 12

Setup for scATAC-seq library purification via QIAquick columns with extender tubes

Figure 13

Single-cell genotyping procedures Liquid handling platforms used at individual steps, as well as volumes transferred, are indicated next to the arrows. A schematic overview of the amplification and barcoding strategy at each step is shown below.

Figure 14

Mosquito setup for genotyping PCR2 plate pooling (A) Schematic overview of the procedure for pooling genotyping PCR2 plates. (B) Mosquito program for genotyping PCR2 plate pooling. (C) Setup of plates on the Mosquito.

Figure 15

Quality control of genotyping libraries (A) TapeStation bands showing genotyping amplicons for each plate (382 pooled cells). Amplicons of expected size should be observed. (B) A TapeStation trace showing intensity of signal as function of size in bp.

Figure 16

Sequencing strategy for genotyping libraries CS2rc is the reverse complement of the CS2 sequence. Note that, if sequencing on the NextSeq, a longer version of the CS1 primer (LCS1) is used instead of the CS1 primer.

Figure 17

Bioanalyzer traces showing ATAC libraries when 10, 15, or 20 genomic loci were co-amplified Human CD34+ bone marrow cells were used. Each trace derives from 96 pooled single cells.

Figure 18

Representative suboptimal ATAC traces (A)–(I) Bioanalyzer traces displaying failed or suboptimal ATAC library amplification, which requires troubleshooting.