Next-generation interaction screening to discover luciferase (construct #1) related protein-protein interactions regulating barley powdery mildew disease immunity and susceptibility

This luciferase (construct # 1) dataset was part of a larger Y2H screen (GEO GSE150396) to identify novel proteins involved in barley immune responses. Yeast-two-hybrid (Y2H) screening was coupled with next-generation sequencing to identify and quantify interacting proteins. The luciferase (construct # 1) bait was mated to a cDNA prey library derived from a 0-48 hour time course of infected leaf tissue. Screens were performed in batch liquid culture to enrich yeast populations for cells expressing positive interactions. After two rounds of enrichment under selective (Histidine absent) and non-selective (Histidine present) conditions, yeast cells were collected. Y2H plasmids were extracted and prey cDNA amplicons were generated via low-cycle PCR. Fragmented amplicons were used as input to generate sequencing libraries and processed on the HiSeq 3000 platform. Reads were mapped to the barley and Blumeria graminis genomes and read counts were analyzed using a custom data processing and scoring pipeline. Putative interactors were cloned and binary Y2H was used to confirm interactions. Y2H screening of a cDNA library was coupled to next-generation sequencing to identify and rank positive protein-protein interactions. Screening for the luciferase (contruct # 1) bait was conducted in triplicate with yeast cells grown in selective and non-selective conditions. NuGEN’s AnyDeplete technology was used to deplete HORVU3Hr1G072260 (zinc finger protein) from the samples. This dataset represents one of two luciferase constructs that were used as bait negative controls. Samples were run on three lanes of Illumina’s HiSeq 3000.

本荧光素酶（构建体1号）数据集隶属于一项更大规模的酵母双杂交（Yeast-two-hybrid，简称Y2H）筛选实验（对应基因表达综合数据库（Gene Expression Omnibus，GEO）收录编号GSE150396），旨在挖掘参与大麦免疫应答的新型蛋白质。该酵母双杂交筛选结合了下一代测序技术，用于鉴定并定量分析互作蛋白质。以构建体1号荧光素酶作为诱饵蛋白，与源自感染叶片组织0-48小时时间序列的cDNA猎物文库进行杂交配对。筛选实验采用批量液体培养方式，以富集表达阳性互作蛋白的酵母细胞群体。在选择性（不含组氨酸）与非选择性（含组氨酸）条件下完成两轮富集后，收集酵母细胞。提取酵母双杂交质粒，并通过低循环PCR扩增获得猎物cDNA扩增子。将扩增子片段化后作为起始原料构建测序文库，并在HiSeq 3000测序平台上完成测序处理。将测序读段比对至大麦与禾本科布氏白粉菌（Blumeria graminis）基因组，随后通过定制化数据处理与打分流程对读段计数进行分析。对推定的互作蛋白进行克隆，并采用二元酵母双杂交技术验证其互作关系。本次cDNA文库的酵母双杂交筛选结合下一代测序技术，用于鉴定并排序阳性蛋白质-蛋白质互作关系。针对构建体1号荧光素酶诱饵的筛选实验设置了三次生物学重复，酵母细胞分别在选择性与非选择性条件下培养。使用NuGEN公司的AnyDeplete技术对样本中的HORVU3Hr1G072260（锌指蛋白）进行了去除处理。本数据集为用作诱饵阴性对照的两款荧光素酶构建体之一。测序样本在Illumina HiSeq 3000平台的三个泳道上完成测序。