3-D human epidermal tissue cultures bulk RNA-Seq expression

This dataset corresponds to gene expression changed occurring in the formation of 3-D human epidermal raft cultures. Normal human epidermal keratinocytes were isolated from epidermis (n=3) and grown using J2-3T3 mouse fibroblasts as a feeder layer originally described by Rheinwald and Green53. 3-D human epidermal raft cultures seeded in collagen hydrogels were prepared using three distinct donor pools as described previously54 and grown at an air-liquid interface for 12 days in E-Medium (DMEM/DMEM-F12 (1:1), 5% Fetal Bovine Serum, adenine (180µM), Bovine pancreatic insulin (5µg/ml), Human apo- transferrin (5µg/ml), triiodothyronine (5µg/ml), L-Glutamine (4mM), Cholera toxin (10ng/ml), Gentamicin (10µg/ml), Amphotericin B (0.25µg/ml)). At day 9 at an air-liquid-interface to allow for epidermal maturation, the epidermal rafts (RHE) were treated with 0.1% BSA/phosphate-buffered saline (Sigma Aldrich, St Louis, MO) for 72 Hrs. Epidermal tissues were separated at the stages from Sub-confluent stage to 3-D raft on day 12 (Sub-confluent, Day 0-Confluent, Day 3-Confluent, Day 3-Raft, Day 6-Raft, Day 9-Raft, Day 12-Raft) from the collagen scaffold and lysed in QIAzol for RNA isolation. RNA samples were sent to the University of Michigan Advanced Genomics Core for RNA sequencing. Libraries for RNA-Seq were generated from polyadenylated RNA and sequenced at six libraries per lane on the Illumina Genome Analyzer IIx. We used Tophat2 to align RNA-seq reads to the human genome, using annotations of GENCODE as gene model. HTSeq was used to quantify gene expression levels. Normalization was performed by DESeq2.

本数据集对应三维人表皮筏状培养（3-D human epidermal raft cultures）形成过程中发生的基因表达变化。从表皮中分离得到正常人生表皮角质形成细胞（normal human epidermal keratinocytes），样本量n=3，并以J2-3T3小鼠成纤维细胞（J2-3T3 mouse fibroblasts）作为饲养层（feeder layer）进行培养，该培养体系最初由Rheinwald与Green于文献53中报道。将接种于胶原水凝胶（collagen hydrogels）中的三维人表皮筏状培养物，采用3个不同供体池（donor pools）进行制备，具体方法参照此前文献54的报道；随后将其置于气液界面（air-liquid interface），使用E培养基（E-Medium，成分为DMEM/DMEM-F12按1:1混合、5%胎牛血清（Fetal Bovine Serum）、180μM腺嘌呤（adenine）、5μg/ml牛胰腺胰岛素（Bovine pancreatic insulin）、5μg/ml人运铁蛋白（Human apo-transferrin）、5μg/ml三碘甲状腺原氨酸（triiodothyronine）、4mM L-谷氨酰胺（L-Glutamine）、10ng/ml霍乱毒素（Cholera toxin）、10μg/ml庆大霉素（Gentamicin）、0.25μg/ml两性霉素B（Amphotericin B））培养12天。在气液界面培养至第9天以促进表皮成熟时，向表皮筏状培养物（epidermal rafts，RHE）施加0.1%牛血清白蛋白（BSA）/磷酸盐缓冲液（phosphate-buffered saline，Sigma Aldrich，美国密苏里州圣路易斯市）处理72小时。从胶原支架上分离处于不同发育阶段的表皮组织，阶段覆盖从亚汇合期（Sub-confluent stage）至第12天的三维筏状培养，具体包括亚汇合期、第0天汇合期、第3天汇合期、第3天筏状培养、第6天筏状培养、第9天筏状培养及第12天筏状培养；随后将组织在QIAzol试剂（QIAzol）中裂解以进行RNA提取。将RNA样品送至密歇根大学先进基因组学中心进行RNA测序。从聚腺苷酸化RNA（polyadenylated RNA）中构建RNA测序文库，随后在Illumina Genome Analyzer IIx测序平台上以每泳道6个文库的方式进行测序。我们采用Tophat2工具将RNA测序读段比对至人类基因组，以GENCODE基因注释集（GENCODE）作为基因模型；使用HTSeq工具对基因表达水平进行定量，并通过DESeq2工具完成数据标准化。