2100 expert

Total RNA from fresh HSCs or cultured HSCs was isolated by Allprep DNA/RNA Micro Kit (Qiagen). Integrity of RNA was validated by Bioanalyzer (2100 Expert, Agilent). Gene expression analysis was undertaken using the HumanHT-12 v4 Expression BeadChip (illumina). The data were 2-based logarithm-transformed and the linear mixed model was used to reveal the overall association between the abundance of mRNA and conditions used to culture HSC. If the overall association was significant (p-value < 0.01), then pairwise comparisons using Tukey’s post hoc tests were performed to detect the difference in expression between treatments. An adjusted p-value < 0.01 and fold change > = 2 or < = −2 were considered statistically significant. PCA was used to visualize the similarities and variations among samples. Sample clustering and heat map analyses were performed using R (version 3.0.2, R Foundation) and the program dChip26 (link). We deposited the data at the Gene Expression Omnibus (GEO) public repository under the accession number GSE67093.

In the library preparation, after clean up (step 16 in Nextera XT DNA Sample Preparation Guide from January 2016, page 14), fragment sizes were measured on a BioAnalyzer (Agilent Technologies) using a high-sensitivity DNA chip and 1 μL of the library. Data were analyzed using the 2100 Expert software version B.01.03 (Agilent Technologies). The correlation area was calculated between 200 and 1000 bp.

Hair roots (approximately 2–3 mm) were used as the source for extracted mRNA. The roots were cut into approximately 15 fragments (0.1–0.2 mm each) by using a microsurgical knife under a stereoscopic microscope. The collected fragments were immersed in 800 μl of ISOGEN reagent (Nippon Gene; Toyama, Japan) in tubes and stirred (15 sec × 2 times) using a Bioruptor UCD-250 sonication device (Cosmo Bio; Tokyo, Japan). Next, the RNA was purified from hair lysates using an ISOGEN kit according to the manufacturer’s instructions. Briefly, the tubes were maintained at room temperature for 5 min, followed by the addition of 200 μl of chloroform. The subsequent process of RNA purification was performed according to the manufacturer’s instructions. After isolation, RNA pellets were washed with 70% ethanol, air dried, and resuspended in 10 μl of RNA-free water (Gibco-BRL; Gaithersburg, MD). Total RNA was quantified at 260 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.; Wilmington, DE). RNA quality was determined using an Agilent Bioanalyzer 2100 (Agilent Technologies; Palo Alto, CA). The 28S:18S rRNA ratio and the RNA integrity number (RIN) were calculated using the 2100 Expert and RIN Beta Version software (Agilent Technologies), respectively.