【Fast ATAC protocol】减少线粒体DNA干扰(wet-2)

参考原文：
http://www.nature.com/ng/journal/v48/n10/full/ng.3646.html#methods

Fast-ATAC sequencing.

This protocol has been optimized for blood cells. We note that digitonin is a gentle detergent and this protocol may not be ideal for cell lines and other cell types that are more resistant to lysis. Five thousand sorted cells in FACS buffer were pelleted by centrifugation at 500g RCF for 5 min at 4 °C in a precooled fixed-angle centrifuge. All supernatant was removed using two pipetting steps, being careful to not disturb the cell pellet, which was not visible. Fifty microliters of transposase mixture (25 μl of 2× TD buffer, 2.5 μl of TDE1, 0.5 μl of 1% digitonin, and 22 μl of nuclease-free water) (FC-121-1030, Illumina; G9441, Promega) was added to the cells, and the pellet was disrupted by pipetting. Transposition reactions were incubated at 37 °C for 30 min in an Eppendorf ThermoMixer with agitation at 300 rpm. Transposed DNA was purified using a QIAGEN MinElute Reaction Cleanup kit (28204), and purified DNA was eluted in 10 μl of elution buffer (10 mM Tris-HCl, pH 8). Transposed fragments were amplified and purified as described previously48
with modified primers23
. Libraries were quantified using qPCR before sequencing. All Fast-ATAC libraries were sequenced using paired-end, dual-index sequencing on a NextSeq instrument with 76 × 8 × 8 × 76 cycle reads.

ATAC-seq data analysis.

ATAC-seq data were processed as previously described23
with notable exceptions. In brief, reads were trimmed using a custom script and aligned using Bowtie 2. To call peaks, data were aggregated by each unique cell type, and peak summits were called using MACS2 and filtered using a custom blacklist, as previously described23
.
To generate a non-redundant list of hematopoiesis- and cancer-related peaks, we first extended summits to 500-bp windows (±250 bp). We then ranked the 500-bp peaks by summit significance value (defined by MACS2) and chose a list of non-overlapping, maximally significant peaks. The complete data set comprised a total of 590,650 peaks. To annotate peaks with promoter and distal labels and the nearest gene, we used the HOMER package with command annotatePeaks.pl. As described previously23
, we counted fragments for each sample across all 590,650 peaks to provide a count matrix. To obtain normalized fragment counts, which were used for all downstream processing, we first performed quantile normalization followed by GC content normalization (CQN R package47
). Data tracks, used solely for visualization, were normalized to the number of fragments falling within all peaks for each sample. Coverage tracks were visualized using the Gviz R package. Fragment yield (Supplementary Fig. 1e) was computed by multiplying the library diversity calculated using Picard tools by the number of reads falling within peaks; values were then divided by the number of cells used in each assay.
For information on transcription factor–based analyses, see the Supplementary Note.

目前感觉： Fast protocol >Ido protocol> standard protocol.