scATAC_Methods

参考文献：Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion

是Nature technology上的文章，作者是华人科学家Howard Chang：https://doi.org/10.1038/s41587-019-0206-z

Methods

Cell lines and PBMC/bone marrow samples

样本制备中，作者为了衡量单细胞捕获效率和multiplets率，选择将人和鼠的样本混合制备；如果是multiplets，那么这个“细胞”对应barcode的序列就会同时比对到hg19和mm的基因组上

For monocyte and T cell mixing experiments, nuclei were first extracted and transposed, then mixed at indicated ratios. To avoid pipetting errors, a large
number of nuclei were mixed after nuclei extraction and transposition, and a smaller number of nuclei were loaded onto the microfluidics chip for scATAC library generation.

multiplets.jpg

BCC sample collection and cell sorting

文章作者希望比较免疫治疗（immunotherapy (PD-1 blockade)）前后患者（basal cell carcinoma，BCC）肿瘤组织微环境的变化：

Singlecell deconvolution of the tumor microenvironment (TME) revealed distinct types of immune, stromal and malignant cells, and analysis of intratumoral T cells identified regulators of therapy-responsive T cell subtypes, including CD8+ exhausted (TEx) and CD4+ T follicular helper (Tfh) cells.

为了尽可能地减少非免疫疗法造成的微环境的变化，作者排除了那些先前有接受过检查点阻断疗法的患者，或者曾接受过免疫抑制剂处理的患者

To minimize non-therapy-related immune cell variation, we excluded patients with previous exposures to checkpoint blockade, or to systemic immune suppressants within 4 weeks of biopsy.

scATAC-seq using the 10x Chromium platform

单细胞ATAC-seq的protocol参考10x官网

All protocols to
generate scATAC-seq data on the 10x Chromium platform, including sample preparation, library preparation and instrument and sequencing settings, are described below and are also available here:

https://support.10xgenomics.com/single-cell-atac

Nuclei isolation

参考的protocol是：Nuclei Isolation
for Single Cell ATAC Sequencing (10x Genomics)

初始细胞数：100,000 to
1,000,000 cells

Nuclei
were centrifuged (500g for 5 min at 4 °C) and the supernatant removed without disrupting the nuclei pellet.

细胞核重悬后的浓度：5,000–7,000 nuclei per µl
based on the starting number of cells

对于细胞数较少的样本，细胞核分离的protocol需要进行调整：裂解buffer和重悬buffer的体积

For low-cell-number BCC samples (less than 20,000 cells), 2 modifications were made to the nuclei isolation protocol. First, 50 µl chilled Lysis Buffer was used instead of 100 µl chilled Lysis Buffer. Second, isolated nuclei were resuspended in 7 µl chilled Diluted Nuclei Buffer; 2 µl was used for cell counting, and 5 µl was used in the downstream library construction protocol

Library construction

scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics; CG000168 Rev B).

对细胞核进行counting后，核的浓度根据the number of available nuclei和desired multiplet rate进行调整；核的浓度不能太低也不能太高，太低了数据量不够，太高了会有multiplets（一个GEM里多于一个细胞核）作者建议：To minimize potential multiplets, we typically aimed to capture <6,000 nuclei per channel

Approximately 100,000 GEMs are formed in each channel (8 channels per microfluidic chip)，and approximately 80% of GEMs contain a single gel bead. Gel beads oligos were newly designed to consist of a 29-bp sequencing adapter, a 16 bp barcode selected from ~750,000 designed sequences (to index droplets) and the first 14 bp of read 1N (primers of the linear amplification reaction)

文库构建：先是线性扩增产物引入barcode和P5接头，然后GEM破裂释放DNA，这些DNA混合起来进行第二轮PCR扩增引入sample index和P7接头

inside individual GEM.jpg Pooled Amplified DNA Processed in Bulk.jpg