Truseq small rna prep kit

Extract RNA reagent (Evrogen, Russia) was used for total RNA extraction from adult 5 days old males. To obtain the fraction of small RNA, ~25 μg of total RNA were separated using 15% polyacrylamide gel electrophoresis in the presence of Urea (8 M) following excision of small RNA fraction corresponding to 21–29 nts. Illumina TruSeq Small RNA prep kit (Illumina, USA) was used for mall RNA libraries preparation. Sequencing was done on an Illumina HiSeq 2000 platform.

RNA was isolated using Trizol (Life Technologies), and 1 μg of total RNA was used to generate small RNA libraries using the TruSeq Small RNA Prep Kit (Illumina). miRNA expression was found with MirDeep2 [41 (link)] and Bowtie [42 (link)] (hg19), using miRBase version 21 [43 (link)]. Read counts from miRNA families (miRNAs with the same seed sequence) were combined.

We determined the circulating miRNA profile in the procured human plasma specimens by performing the HTG EdgeSeq miRNA Whole Transcriptome Assay (miRNA WTA) (HTG Molecular Diagnostics, Inc. Tucson, AZ). The HTG EdgeSeq miRNA WTA enables us to measure the expression of 2,083 human miRNA transcripts using next-generation sequencing (NGS). The plasma samples (20 µl) were incubated with 20 µl plasma lysis buffer plus 4 µl of proteinase-K (miRNA lysis buffer kit; HTG Molecular Diagnostics) with constant shaking at 50֯C for 3 h to denature them. The miRNA expression profile was determined as per standardized protocols [6 (link)]. Briefly, the samples were loaded into an HTG Edgeseq Processor. After the automated preparation process, libraries were prepared with TruSeq Small RNA Prep kit (Illumina). Single-end reads of 51 bp in length were sequenced using an Illumina GAIIx instrument. To quantify the expression levels, trimmed reads were mapped to the genome and duplications were removed. Finally, the annotation from miRBase v20 was used to designate reference mapped reads to mature miRNAs.

We generated and analyzed small RNA (sRNA) libraries from different organs and developmental stages. Asparagus officinalis samples were collected from a commercial field in the T.S. Smith and Son’s Farm (http://www.tssmithandsons.com/), Bridgeville, Delaware, as well as from female, male, and supermale whole spear tip tissue from a previous study^{27 (link)}. Samples were collected and anthers were dissected using a 2 mm stage micrometer (Wards Science, cat. #949910) in a stereo microscope, and immediately frozen in liquid nitrogen until total RNA isolation was performed. Total RNA was isolated using the PureLink Plant RNA Reagent (ThermoFisher Scientific, cat. #12322012) following the manufacturer’s instructions. Small RNAs (20–30 nt) were size selected in a 15% polyacrylamide/urea gel and used for small RNA library preparation using the Illumina TruSeq Small RNA Prep Kit. Detailed description of analysis can be found in Supplementary Note 4.

Back skin of four 8 weeks CD1 mice was trypsinized overnight at 4°C. The epidermis was scraped and chopped into fine pieces to further trypsinize to isolate single cells. The cells were washed with PBS and resuspended in PBS + 1% FBS. Cells were stained for 1 h on ice with integrin α6‐PE (ab95703, Abcam) and/or CD34‐FITC‐conjugated (11‐0341‐85, eBioscience) antibodies. Cells were sorted on FACS Aria™ (BD Biosciences) based on CD34⁺α6⁺ (bulge stem cells), CD34⁻α6⁺ (basal epithelia), and CD34⁻α6⁻ (differentiated keratinocytes; Appendix Fig S1). This was followed by RNA extraction and small RNA library prep (TruSeq Small RNA Prep Kit, Illumina). Small RNA libraries were sequenced on Illumina NextSeq 500 platform.
MDA‐MB‐231 and HS578T breast cancer cells were sorted for CD44/CD24 expression with Alexa Fluor 488‐conjugated α‐CD44 (Stem Cell, Clone IM7, Lot #SC02931) and α‐CD24 (Santa Cruz Biotechnology, FL‐80, SC‐11406 Lot #E1413). Cells were sorted with the FACS Aria™ Fusion Sorter (BD Biosciences; Appendix Figs S2 and S3) operated by the A*STAR SIgN common FACS facility. Each sample was analyzed with negative controls and single channel/color controls.

The TruSeq Small RNA Prep Kit (RS-122-2002, Illumina, USA) was used to construct libraries according to the manufacturer’s protocol. The optimal loading amount was selected for sequencing on the Illumina platform.

Prior to polysome profiling, cells were translationally arrested by treating with CHX for 30 min. Lysates made from various states of differentiation (stem: Wnt3a+LIF‐ and LIF‐treated; differentiating: RA‐ and Wnt3a+RA‐treated) were ultracentrifuged on a 15–45% sucrose gradient at 39,000 rpm to separate the ribosomal fractions. The individual ribosomal subunits (40S, 60S), monosomes (80S), and polysomes were fractionated based on the profile generated by the absorbance of the gradient at 254 nm. The RNA was precipitated from the sucrose gradient collected by ethanol precipitation. The pellets were dissolved in TRIzol, and RNA was isolated from the pellets. Small RNA libraries were prepared from isolated RNA using TruSeq Small RNA Prep Kit (RS200012, Illumina). For statistics, samples treated with Wnt3a+LIF and LIF were grouped as proxies for stem, and RA‐ and Wnt3a+RA‐treated (absence of LIF) as proxies for differentiating state. The data were analyzed as described in Methods Section “Identification and characterization of tsRNAs”. We used Wilcox's test 46 to calculate significance. The change in the tsRNA profiles was calculated using change point analysis (R package: changepoint) 61.