Nebnext small rna library prep kit

After the MDA-MB-231-IV2-1 cells were transfected with si-LOC550643 and the scrambled control for 48 h, the total RNA was extracted from two samples by using TRIZOL reagent. The small RNA library was prepared using the NEBNext small RNA library prep kit (New England Biolabs). The library preparation process is described in details in our previous study (Tseng et al., 2017 (link)). Finally, the small RNA profiles of the MDA-MB-231-IV2-control and MDA-MB-231-IV2-LOC550643 knockdown were performed using the MiSeq V2 reagent kit (150 cycles; Illumina, San Diego, CA, United States). The sequencing data were analyzed using our own tool (Pan et al., 2014 ). All microarray raw data were deposited in the NCBI GEO, and they are all accessible (accession number: GSE175514).

Libraries were constructed using NEBNext Small RNA Library Prep Kit (NEB). Input total RNA was ligated with 3′ adapters, and the excess of 3′ adapter was hybridized with a reverse transcription primer prior to the ligation of the 5′ adapter. Ligated RNA was converted into cDNA with a first strand reverse transcriptase, followed by PCR amplification. Library size was then selected by loading the PCR product into a 3% agarose dye-free gel of a Pippin Prep system (Sage Science). The libraries were 100 bp single-end sequenced. The metrics for all of the library sequencing performed in this project are shown in Supplementary Data 7.

Small RNAs (17–25 nt size) from 20 $\mathsf{\mu }$ g total RNA were size selected by gel extraction using a 17.5% urea acrylamid mini-gel, visualized with SYBR-Gold (Life-Technologies, Darmstadt, Germany). Gel slices were cut and RNA was eluted by overnight incubation in 3 Vol. using 0.3M NaCl at 4 ${}^{\circ }$ C, RNA was precipitated using 3 Vol. Ethanol (100%) and Glycogen (70 ng/ $\mathsf{\mu }$ L) and afterwards subjected to library preparation using the NEBNext small RNA library prep Kit (New England Biolabs, Frankfurt a.M., Germany), according to the manufacturer’s instructions (3 ${}^{\prime }$ -adapter ligation extended to 18h at 16 ${}^{\circ }$ C). Sequencing was done on the Illumina HiSEQ 2500 platform (Illumina, San Diego, CA, USA) using the RAPID mode and 30 nt read length. Reads (1.9; 6.7; 5.5 million Mac mapping reads, respectively) were de-multiplexed and adapter sequences were trimmed using Trim Galore [20 ] that uses Cutadapt [21 (link)] with a stringency cutoff of 10. SmallRNA read alignments to transgenes and normalization of reads were carried out precisely as described in [17 (link)]. The complete analysis was done using the RAPID pipeline [22 ].

Ribosomal RNA-depleted RNA-seq libraries were prepared from 400–600 ng of total RNA using the low input ScriptSeq Complete Gold Kit (Epicentre). For small RNA-seq, RNA was isolated with QIAzol (QIAGEN) and libraries prepared from 400–600 ng of RNA using the NEBNext small RNA library prep kit (NEB), followed by size selection (~120–150 bp, including adaptors) using gel extraction. Libraries were sequenced on an Illumina NextSeq 500 with single-end 75 bp reads.

Tissue and cell samples were dissolved in Isogen (Nippon gene), and total RNAs were prepared by Direct-zol RNA prep kit (Zymo Research). Although other RNA preparation methods are also applicable, we suggest avoiding RNA degradation to capture full-length RNAs. RNA (500 ng) was treated with 25 U of RNA 5′ pyrophosphohydrolase (RppH, New England Biolab) for 1 h at 37 °C in a 50-μl reaction mixture containing 1× Thermopol buffer (New England Biolab). This procedure freed 5′ cap or triphosphate into monophosphate, which can be later ligated by an RNA ligase. The reaction was stopped by phenol-chloroform extraction and ethanol precipitation. Sequencing libraries were constructed by using NEBNext Small RNA Library Prep kit (New England Biolab) according to manufacturer’s instructions. PCR products were run on a 6% native polyacrylamide gel, and a gel region corresponding to 240–380 bp (an insert size of 113–253 bp) was extracted. DNAs were eluted in 300 μl TE, ethanol precipitated, and dissolved in 20 μl of 0.1× TE. Library concentrations were determined by real-time RCR using the KAPA library quantification kit (KAPA Biosystems) on a StepOnePlus (Thermofisher).

For RNA-seq analysis, total RNA was extracted from Myo-EVs, primary mouse bone marrow cells and osteoblasts with TRIZOL reagent (Thermo Fisher Scientific, Inc) and assessed by an Agilent Bioanalyzer with the RNA 6000 Pico Kit (Agilent, Santa Clara, CA, USA). Sequencing library was prepared using the NEBNext Small RNA Library Prep Kit (E7330, New England Biolabs, Ipswich, MA, USA). Total RNA (90 ng) was ligated to 3’ Adaptor, and excess adaptor was subsequently absorbed. RNA fragments were further ligated to 5’ Adaptor for cDNA synthesis. Synthesized cDNA was amplified by PCR for 12 cycles. Small-RNA library was size-selected by AMPure beads (NC9933872, Thermo Fisher Scientific, Inc) and verified with the Bioanalyzer DNA High-sensitivity Kit (5067–4626, Agilent). For obtaining 2 × 36-base paired-end reads, sequencing was performed by the Illumina NextSeq500 (Illumina, San Diego, CA, USA). FASTQ files were imported to CLC Genomics Workbench (Ver.10.1.1, Qiagen, Germantown, MD, USA). Reads were grouped by sequence and the read groups were matched to miR-base annotated micro RNAs. Raw counts of each sample were converted by total count 1,000,000 at first, and the converted counts were further normalized by scaling method with means as normalization values, median mean as reference and trimming percentage 5 for comparing multiple samples.