Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues. Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions are recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) “Calling Cards” into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high performance computing environment and to conduct downstream analyses. Overall design: Calling Cards were transcranially injected with AAV-Calling Cards at P1. Tissue was harvested at P21 and the cortex, midbrain, and hindbrain regions were dissected. Calling Card libraries were prepared to analyze BRD4-binding across the different brain regions.

“呼叫卡技术（Calling Cards）”是一类平台型技术，可在经遗传靶向的细胞类型的基因组中，记录瞬时蛋白质-脱氧核糖核酸（deoxyribonucleic acid, DNA）相互作用的累积历史。此类相互作用的记录可通过下一代测序（next generation sequencing, NGS）技术获取。相较于其他仅能在样本采集时提供瞬时快照式结果的基因组检测技术，呼叫卡技术可实现历史分子状态与最终表型或结局之间的关联分析。
为实现这一目标，呼叫卡技术借助piggyBac转座酶（piggyBac transposase）将自我报告转座子（self-reporting transposon, SRT）“Calling Cards”插入基因组，从而在相互作用位点留下永久性标记。该技术可应用于多种体外（in vitro）和体内（in vivo）生物学系统，用以研究发育、衰老及疾病相关的基因调控网络。其原生版本可直接用于评估增强子的使用活性，同时也可通过定制转录因子（transcription factor, TF）-piggyBac融合蛋白，实现特定转录因子结合位点的谱分析。
呼叫卡技术的实验流程主要包含五个核心步骤：试剂递送、样本制备、文库制备、测序及数据分析。本文首先提供了一套全面的实验设计、试剂选择指南，以及针对该平台的可选定制方案，用以研究更多类型的转录因子（TF）。随后，本文针对上述五个步骤提供了更新后的实验方案，采用了可提升通量、降低成本的新型试剂，并概述了一套新近搭建的计算分析流程。本方案面向具备基础分子生物学实验经验的用户，可在1至2天内完成从样本到测序文库的制备流程。若需在高性能计算环境中搭建该分析流程并开展后续数据分析，则要求使用者具备生物信息学分析及命令行工具的使用经验。
实验整体设计：于出生后第1天（Postnatal day 1, P1）经颅注射腺相关病毒（adeno-associated virus, AAV）-Calling Cards载体以递送呼叫卡技术；于出生后第21天（Postnatal day 21, P21）采集组织，分离大脑皮层、中脑及后脑区域；构建Calling Cards文库，以分析不同脑区中溴结构域蛋白4（BRD4）的结合情况。