Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development

RNA-seq analysis of developmental stages from the filamenous ascomycete Sordaria macrospora. Using laser capture microdissection, we separated protoperithecia (young fruiting bodies) from the surrounding hyphae. RNA isolation and amplification from 150 protoperithecia yields enough material for RNA-seq analysis. The resulting data were compared to RNA-seq data from whole mycelial exctracts (total vegetative and total sexual mycelium) to characterize the genome-wide spatial distribution of gene expression during sexual development. We analyzed total vegetative mycelium, total sexual mycelium, and protoperithecia from the wild type as well as protoperithecia from the sterile mutant pro1. Additionally, we used the RNA-seq information to improve the predicted S. macrospora gene models, and annotated UTRs for more than 50 % of the genes. Overall design: 8 samples were sequenced (2 independent biological replicates for the four conditions "wt sex: SM2, SM7", "wt veg: SM1, SM6", "wt proto: SM4, SM5", "pro1 proto: SM8, SM9", each on one Illumina/Solexa lane (GAII or HiSeq, single reads of 36 or 100 bases))

本数据集针对丝状子囊菌（filamentous ascomycete）大孢粪壳菌（Sordaria macrospora）的发育阶段开展RNA测序（RNA-seq）分析。研究采用激光捕获显微切割（laser capture microdissection）技术，从周边菌丝中分离得到原生子囊壳（protoperithecia，即幼小子实体）。从150个原生子囊壳中提取并扩增RNA，可获得满足RNA测序分析所需的足量样本材料。将本研究所得测序数据与全菌丝提取物（涵盖总营养菌丝与总有性子菌丝）的RNA测序数据进行比对，以解析有性发育过程中基因表达在全基因组范围内的空间分布特征。本研究分析了野生型的总营养菌丝、总有性子菌丝与原生子囊壳，以及不育突变体pro1的原生子囊壳样本。此外，本研究借助RNA测序数据优化了大孢粪壳菌的预测基因模型，并为超过50%的基因注释了非翻译区（untranslated region, UTR）。实验设计概述：共对8个样本完成测序，4组实验条件分别为：野生型有性阶段（"wt sex: SM2, SM7"）、野生型营养阶段（"wt veg: SM1, SM6"）、野生型原生子囊壳（"wt proto: SM4, SM5"）以及不育突变体pro1的原生子囊壳（"pro1 proto: SM8, SM9"），每组设置2个独立生物学重复；每个样本均在单条Illumina/Solexa测序泳道（采用GAII或HiSeq平台）进行测序，获得长度为36或100碱基的单端reads。