Cellulose-induced regulation of Phanaerochaete chrysosporium genes

Transcript profiles of Phanerochaete chrysosporium grown on different substrates were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in cellulose degradation. Keywords: gene expression under different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from below mentioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.

本研究对在不同底物上培养的黄孢原毛平革菌（Phanerochaete chrysosporium）的转录本谱进行了分析。芯片设计基于美国能源部（Department of Energy, DoE）联合基因组研究所（Joint Genome Institute, JGI）v2.1版注释信息，研究目标为明确参与纤维素降解过程的基因。关键词：不同培养条件下的基因表达。

该数据集包含10004个独特等位基因，每个罗氏NimbleGen（Roche NimbleGen，美国威斯康星州麦迪逊市）芯片为每个基因设计了12条独特的60聚体探针，且每组探针均设置三次重复。（仅有2至11条60聚体探针可用于7个主要由重复DNA组成的基因模型。）

从下述培养基中纯化总RNA。简言之，通过Miracloth（Calbiochem，EMD Biosciences，美国新泽西州吉布斯敦市）过滤收集培养物，挤压沥干后置于液氮中快速冷冻。菌体沉淀于-80℃储存备用。

提取缓冲液的配制方法为：将10 mL 690 mM对氨基水杨酸（钠盐）（Sigma-Aldrich，美国密苏里州圣路易斯市）与10 mL 56 mM三异丙基萘磺酸钠（Sigma-Aldrich）混合，置于冰上。向其中加入5 mL 5X RNB缓冲液（1.0 M Tris、1.25 M NaCl、0.25 M EGTA），使用NaOH将5X RNB的pH调至8.5。混合液置于冰上，使用前充分振荡混匀。

将冷冻的真菌菌体沉淀置于经酸清洗并预冷的研钵和研杵中，用液氮研磨成细粉。将研磨后的菌丝转移至Falcon 2059离心管（VWR International，美国宾夕法尼亚州西切斯特市），加入提取缓冲液制成浓稠匀浆。将样品剧烈涡旋后置于冰上，直至所有样品处理完毕。

向每个样品中加入半体积TE饱和苯酚（Sigma-Aldrich）和四分之一体积氯仿（Sigma-Aldrich），剧烈涡旋。将样品置于固定角转子中以2940×g离心5分钟。将水相转移至新离心管，重复酚:氯仿抽提操作，直至水相与有机相之间的界面清晰透明。

将最终的水相转移至洁净的2059离心管中，加入0.1体积pH 5.2的3M乙酸钠（经DEPC处理）和2体积无水乙醇，剧烈振荡后于-20℃过夜储存。将离心管以2940×g离心1小时，弃去上清液，将沉淀重悬于4 mL无RNase水中。使用RNeasy Maxi试剂盒（Qiagen，美国加利福尼亚州瓦伦西亚市），按照制造商提供的RNA纯化方案进行总RNA纯化。通过两次洗脱将RNA从RNeasy离心柱上洗脱，最终体积为2 mL。将洗脱得到的RNA进行乙醇沉淀，于-20℃过夜储存。将RNA以2940×g离心1小时，用70%乙醇洗涤一次，并重悬于50-100 μL无RNase水中。每种培养基设置三次生物学重复，共使用15张独立芯片。

将RNA反转录为双链cDNA，并使用Cy3荧光基团（Cy3 fluorophore）标记样品，由罗氏NimbleGen（冰岛）完成芯片杂交。简言之，取10 μg总RNA，与1×第一链缓冲液、10 mM DTT、0.5 mM dNTPs、100 pM oligo T7 d(T)24引物以及400 U SuperScript II反转录酶（SuperScript II，Invitrogen）混合，于42℃孵育60分钟。通过加入1×第二链缓冲液、0.2 mM dNTPs、0.07 U/μL DNA连接酶（Invitrogen）、0.27 U/μL DNA聚合酶I（Invitrogen）、0.013 U/μL RNase H，于16℃孵育2小时合成第二链cDNA。随后立即加入10 U T4 DNA聚合酶（Invitrogen），于16℃继续孵育5分钟。将双链cDNA用27 ng/μL RNase A（EpiCenter Technologies）处理，于37℃孵育10分钟。使用等体积酚:氯仿:异戊醇（Ambion）纯化经处理的cDNA，经乙醇沉淀、80%乙醇洗涤后，重悬于20 μL水中。

取1 μg每份cDNA样品，使用1 U/μL Klenow片段（Klenow Fragment，New England BioLabs）和1 O.D.单位Cy3荧光基团（TriLink Biotechnologies, Inc.）于37℃孵育2小时进行扩增与标记。将6 μg标记好的cDNA悬浮于NimbleGen杂交液中，于42℃进行17小时的芯片杂交。使用Axon4000B扫描仪（Molecular Dynamics）扫描芯片，通过NimbleScan v2.4软件从扫描图像中提取数据。使用DNASTAR ArrayStar v2.1（美国威斯康星州麦迪逊市）软件对数据进行定量与可视化分析。所有分析均基于每种培养基的三次生物学重复。对整个数据集应用分位数归一化（quantile normalization）与稳健多阵列平均（robust multi-array averaging, RMA）算法进行处理。