Library quantification kit universal

Samples used in the study were collected in Amazonian savanna enclaves: Byrsonima coccolobifolia (voucher BHCB 169523) from Boa Vista (60°49′45′′W, 2°39′40′′N) and B. crassifolia (voucher BHBC 169445) from Alto Alegre (61°09′04′′W, 3°09′45′′N). Voucher specimens were deposited in BHCB herbarium (Herbarium of Departamento de Botânica, Universidade Federal de Minas Gerais). Genomic DNA was extracted from silica-dried leaves, using Novaes et al.^{57 (link)} protocol. DNA quality was assessed in a spectrophotometer Nanodrop 2000 (Thermo Scientific) and integrity was evaluated using a 0.8% agarose gel. In addition, DNA was quantified through fluorometry using Qubit 2.0. (Life Technologies). DNA samples from each species were used to prepare two separate libraries with Nextera kit (Illumina Inc., San Diego, CA), following manufacturer’s protocol. Different barcodes were used to identify DNA fragments derived from each species. To guarantee the intended fragments size, aliquots of each library were ran in 1% agarose gel and quantified by quantitative PCR, using a Library Quantification Kit – Illumina/Universal (Kapa Biosystems Inc., Wilmington, MA). Short fragments of approximately 600 bp from both libraries were combined and submitted for paired-end sequencing using a single lane on a MiSeq sequencer (Illumina Inc.).

Total RNA, including short RNAs, was purified from frozen hepatocytes (pooled 10 donors HMCS10, GIBCO) and cells harvested at two different time points: day 20 of hepatic differentiation (hepatoblast stage of HD) and day 24, the last day of differentiation, using the miRNeasy Micro Kit (Qiagen) and quantified by NanoDrop spectrophotometer. Total RNA was used in the small RNA protocol with the TruSeq™ Small RNA sample prep kit v2 (Illumina) according to the instructions of the manufacturer. The barcoded libraries were size restricted between 140 and 165 bp, purified, and quantified using the Library Quantification Kit Illumina/Universal (KAPA Biosystems) according to the instructions of the manufacturer. Sequencing was performed with an Illumina HiScanSQ sequencer at the sequencing core facility of the IZKF Leipzig (Faculty of Medicine, Leipzig University).

Total DNA from sponge and freshwater samples were sent to the BPI sequencing service. Sponge and freshwater DNA were purified with magnetic beads prior to performing the PCRs. DNA was PCR amplified in triplicate. Reactions contained 0.3 μM of each universal primer for V4 regi on of 16S rRNA (Caporaso et al., 2011 (link)), 1 × buffer GoTaq Colorless Master Mix (Promega), 20 ng of genomic DNA in a total volume of 20 μl. Amplification was performed using the following program: 94°C for 3 min, then 29 cycles of 94°C for 45 s, 50°C for 1 min, and 72°C for 1 min, and a final extension of 72°C for 1 min 30 s. Amplicons were analyzed using 2% agarose gel electrophoresis. After combining the triplicate products, they purified using Agencourt AMPure XP kit (Thermo Fisher Scientific) and quantified by real-time PCR, all according to the manufacturer’s protocol KAPA-KK4824 (Library Quantification Kit – Illumina/Universal, Kapa Biosystems). An equimolar pool of DNA was generated by normalizing all samples at 2 nM for the sequencing, which was conducted using the Illumina MiSeq new generation sequencing system (Illumina^® Sequencing). After sequencing, a FASTQ file containing the sequences was generated.

In total, 500 ng RNA was extracted using TRIzol (ThermoFisher Scientific) method and used for small RNA library preparation with the TruSeqTM Small RNA Sample Prep Kit v2 (Illumina), according to the manufacturer’s instructions. The barcoded libraries were size restricted between 140  and 165 bp purified, and quantified using the Library Quantification Kit–Illumina/Universal (KAPA Biosystems), according to the manufacturer’s instructions. A pool of up to 12 libraries was used for cluster generation per lane. Library DNA at a concentration of 10 pM was clustered using an Illumina cBot, according to the SR_Amp_Lin_Block_Hybv8.0 protocol of the manufacturer. Sequencing of 50 bp was performed with an Illumina HighScan-SQ sequencer using version 3 chemistry and the version 3 flow cell according to the instructions of the manufacturer. Results were aligned to miRBase v21 and normalized. Median expression of each miRNA was set as threshold for low or high expression. Each experimental group was then analyzed for its level of expression compared to the calculated threshold.

For deep sequencing, RNA from MDA-MB-231 cells that were obtained after the different treatments was used in four independent experiments. A total of 500 ng/total RNA was used for library synthesis with the TrueSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The barcoded libraries were purified and quantified while using a Library Quantification kit–Illumina/Universal (KAPA Biosystems) on a TaqMan 7500 Real-Time PCR System. A pool of up to 10 libraries was used for cluster generation at a concentration of 10 nM each using an Illumina cBot. The sequencing of 2 × 100 bp was performed with an IlluminaHighScan-SQ sequencer at the sequencing core facility of the Faculty of Medicine (University Leipzig) using version 3 chemistry and flowcell according to the instructions of the manufacturer.

Total RNA was extracted from 20-50 mg snap-frozen thyroid tissue using 1 mL Trizol reagent. Reverse transcription was done with miScript Reverse Transcription kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols in a 10 μL reaction using 1 μg of total RNA. 500 ng of total RNA prepared from the tumor samples was used in the small RNA protocol with the TruSeq™ Small RNA sample prepkit v2 (Illumina) according to the instructions of the manufacturer. The barcoded libraries were size restricted between 140 and 165 bp, purified and quantified using the Library Quantification Kit - Illumina/Universal (KAPA Biosystems) according to the instructions of the manufacturer.