Transcriptome analysis of basal and stimulated von Willebrand factor release from endothelial colony forming cells derived from Type 1 von Willebrand disease patients

All participants provided informed consent and this study was approved by the Queen’s University Health Sciences Ethics Review Board.
Type 1 VWD patients ≥18 years of age were recruited through Inherited Bleeding Disorders Clinic of Southeastern Ontario in Kingston, Canada. Inclusion criteria for Type 1 VWD patients included a VWF antigen (VWF:Ag) and/or VWF activity (VWF:GPIbM or VWF:RCo) levels between 0.05 and 0.50 IU/mL, a VWF:GPIbM/VWF:Ag ratio >0.6, and normal VWF multimers.

Illumina compatible libraries were constructed using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Austria) using 350ng of total RNA as input. The library size and concentration were determined using the Labchip GX (Perkin Elmer) and Qubit (Thermo Fisher) platforms, respectively. Independently indexed and purified libraries were combined at an equimolar concentration and the library pool was subject to bead purification, denaturation, and dilution. This pooled library was loaded onto a MidOutput v2 reagent cartridge at a concentration of 2.2pM and subject to 75 cycles of single-ended sequencing to a depth exceeding 5 million clusters per sample on a NextSeq550 sequencer (Illumina, California). Raw data in the form of .fastq files were transferred to the Queen’s Center for Advanced Computing, Frontenac cluster, and assessed for quality and trimmed using an established pipeline. Briefly, sequencing reads were aligned to the Ensembl_GRCh38 human genome using STAR aligner and counts were generated using HTSEQ-COUNT.

miRNA profiling was conducted according to an established barcoded small RNA sequencing platform. Briefly, 100ng of purified RNA was spiked with a set of synthetic calibration markers prior to ligation to a sample-specific 3’ oligonucleotide adaption specific for small RNA species. Following sequencing, .fastq files were uploaded through a web-accessible RNA sequencing pipeline (RNAworld.rockefeller.edu) hosted in the Tuschl Laboratory at The Rockefeller University. To report miRNA abundance independent of sequencing depth, read counts were normalized against total sequence reads annotated as miRNAs.

mRNA sequencing raw counts:
File Name New name
MZ01N_pos_S12.counts.txt 1C pos
MZ01P_neg_S1.counts.txt 1P neg
MZ01P_pos_S2.counts.txt 1P pos
MZ02P_neg_S3.counts.txt 2P neg
MZ02P_pos_S4.counts.txt 2P pos
MZ04N_neg_S13.counts.txt 2C neg
MZ04N_pos_S14.counts.txt 2C pos
MZ05P_neg_S5.counts.txt 3P neg
MZ05P_pos_S6.counts.txt 3P pos
MZ06N_neg_S15.counts.txt 3C neg
MZ06N_pos_S16.counts.txt 3C pos
MZ06P_neg_S7.counts.txt 4P neg
MZ06P_pos_S8.counts.txt 4P pos
MZ08P_neg_S9.counts.txt 5P neg
MZ08P_pos_S10.counts.txt 5P pos
MZ10N_neg_S17.counts.txt 4C neg
MZ10N_pos_S18.counts.txt 4C pos
P053_neg_S19.counts.txt 6P neg
P053_pos_S20.counts.txt 6P pos
V447_neg_S21.counts.txt 7P neg
V447_pos_S22.counts.txt 7P pos
V449_neg_S23.counts.txt 8P neg
V449_pos_S24.counts.txt 8P pos

miRNA data
miRNA_processed_data.xlsx

所有参与者均提供了知情同意，且本研究已获得女王大学健康科学伦理审查委员会的批准。第一型血管性血友病（VWD）患者≥18岁者，通过加拿大金斯顿东南安大略省的遗传性出血性疾病诊所招募。第一型VWD患者的纳入标准包括血管性血友病因子抗原（VWF:Ag）及/或血管性血友病因子活性（VWF:GPIbM或VWF:RCo）水平介于0.05至0.50 IU/mL之间，VWF:GPIbM/VWF:Ag比值大于0.6，以及血管性血友病多聚体水平正常。利用Lexogen（奥地利）的QuantSeq 3’ mRNA-Seq Library Prep Kit构建与Illumina兼容的文库，以350ng的总RNA为输入。文库大小和浓度分别采用Labchip GX（Perkin Elmer）和Qubit（Thermo Fisher）平台测定。独立索引和纯化的文库以等摩尔浓度混合，文库池经过磁珠纯化、变性及稀释处理。此混合文库以2.2pM的浓度加载至MidOutput v2反应柱，并经过75个循环的单端测序，在Illumina NextSeq550测序仪上每样本深度超过500万个簇。原始数据以.fastq文件形式传输至女王大学高级计算中心、Frontenac集群，并使用既定流程进行质量评估和剪切。简而言之，测序读段通过STAR aligner对Ensembl_GRCh38人类基因组进行对齐，并使用HTSEQ-COUNT生成计数。miRNA分析依照既定的条形码小RNA测序平台进行。简而言之，100ng纯化RNA在连接至针对小RNA物种的特定3’寡核苷酸适配体之前，与一套合成校准标记物混合。测序后，.fastq文件通过位于洛克菲勒大学Tuschl实验室的在线RNA测序流程（RNAworld.rockefeller.edu）上传。为独立于测序深度报告miRNA丰度，读段计数以注释为miRNA的总序列读段进行标准化。mRNA测序原始计数如下所示：

文件名 新名称

MZ01N_pos_S12.counts.txt 1C正

MZ01P_neg_S1.counts.txt 1P负

MZ01P_pos_S2.counts.txt 1P正

MZ02P_neg_S3.counts.txt 2P负

MZ02P_pos_S4.counts.txt 2P正

MZ04N_neg_S13.counts.txt 2C负

MZ04N_pos_S14.counts.txt 2C正

MZ05P_neg_S5.counts.txt 3P负

MZ05P_pos_S6.counts.txt 3P正

MZ06N_neg_S15.counts.txt 3C负

MZ06N_pos_S16.counts.txt 3C正

MZ06P_neg_S7.counts.txt 4P负

MZ06P_pos_S8.counts.txt 4P正

MZ08P_neg_S9.counts.txt 5P负

MZ08P_pos_S10.counts.txt 5P正

MZ10N_neg_S17.counts.txt 4C负

MZ10N_pos_S18.counts.txt 4C正

P053_neg_S19.counts.txt 6P负

P053_pos_S20.counts.txt 6P正

V447_neg_S21.counts.txt 7P负

V447_pos_S22.counts.txt 7P正

V449_neg_S23.counts.txt 8P负

V449_pos_S24.counts.txt 8P正

miRNA数据

miRNA_processed_data.xlsx