Experiments with NEBNext® (NEB), TruSeq® (Illumina), NEXFlex™ (Bioo Scientific), QIAseq (Qiagen), and SMARTer (Takara Bio) were performed following the manufacturers’ recommendations. For all kits, 1 pmol of the miRXplore™ Universal Reference (Miltenyi Biotec) was used as input to test accuracy in detection (Figs. 2 and 3 ). Brain total RNA (1 μg) was used for Fig. 3 for all kits with the exception of QIAseq, where 500 ng of total RNA was used as per the manufacturer’s recommendations. Libraries were prepared in triplicate for all experiments. To determine concentration and quality of libraries, all libraries were analyzed with an Agilent D1000 ScreenTape on a 2200 TapeStation instrument (Agilent) and then quantified with a Qubit dsDNA BR Assay kit on a Qubit 3.0 instrument.
Agilent d1000 screentape
Manufactured by Agilent Technologies
Sourced in United States
The Agilent D1000 ScreenTape is a lab equipment product designed for automated electrophoresis analysis. It provides a standardized and efficient method for analyzing DNA and RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Tapestation 2200, by Agilent Technologies (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nebnext, by New England Biolabs (1 mentions)
Mirxplore universal reference, by Miltenyi Biotec (1 mentions)
Qiaseq, by Qiagen (1 mentions)
Truseq, by Illumina (1 mentions)
Truseq stranded total rna preparation kit, by Illumina (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Ampure xp beads, by Agilent Technologies (1 mentions)
Agilent high sensitivity d1000 screentape, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
4200 tapestation system, by Agilent Technologies (1 mentions)
Truseq stranded mrna sample prep kit, by Illumina (1 mentions)
Nextseq 500 550 platform, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Truseq rna library prep kit v2, by Illumina (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 3000 4000, by Illumina (1 mentions)
9 protocols using agilent d1000 screentape
1
Comparative Analysis of miRNA Library Prep Kits
2
YTHDF2 Depletion in MYC-ER HMECs
3
Optimized DNA Fragmentation and Library Prep
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DNA of the dilution series was fragmented by sonication using Covaris Sonalab 7.1 S220 [Covaris, Woburn, MA, USA] (80 s, peak power 140.0, duty factor 10.0, cycles/burst 200, power ~ 12, temp below 12 °C). Shearing results were checked by electrophoresis using an Agilent D1000 screen tape [Agilent, Santa Clara, CA, USA]. The mean size of the fragmented DNA was ~300 bp. Sample preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina sequencing [New England Biolabs, Ipswich, MA, USA] using an input amount of 1 µg DNA in 50 µl Tris-EDTA. NEB adapters were substituted for unique molecular identifier (UMI) TruSeq dual-index duplex adapters (15 µM) [Integrated DNA Technologies (IDT), Coralville, IA, USA], and USER enzyme steps were skipped. UMIs are used to remove duplicate reads and reduce the error rate during the data-analysis procedure. A size-selection to 300–400 bp using AMPure XP Beads [Beckman Coulter, Indianapolis, IN, USA] was performed after adding adapters. IDT xGen Library Amplification primers (5 µM p5 and 5 µM p7) were used to enrich the adapter-ligated DNA using PCR (12 cycles). The amplified product was measured by electrophoresis using the Agilent High Sensitivity D1000 screen tape after cleanup with AMPure XP Beads.
4
Targeted-Enrichment DNA Library Preparation
5
Illumina RNA-seq Library Preparation
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cDNA libraries from total RNA samples (n = 40) were prepared using an Illumina TruSeq Stranded mRNA sample prep kit (Illumina, San Diego, CA) following a balanced batch-group design. 3 µg of total RNA were used as the RNA input according to the manufacturer’s protocol. mRNAs were isolated from the total RNAs by purifying the poly-A containing molecules using poly-T oligo attached to magnetic beads. The RNA fragmentation, first- and second-strand cDNA syntheses, end repair, single ‘A’ base addition, adaptor ligation, and PCR amplification were performed according to the manufacturer’s protocol. The average size of the cDNA libraries was approximately 350 bp (including the adapters). cDNA libraries were quantified using an Agilent D1000 ScreenTape in a 4200 TapeStation system (Agilent Technologies Inc). Libraries were normalized to 10 nM and pooled in equal volumes. The pool concentration was quantified by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems Inc) before sequencing in a NextSeq 500/550 cartridge of 150 cycles (Illumina). Indexed and pooled samples were sequenced 150 bp paired-end reads by on the Illumina NextSeq 500/550 platform according to the Illumina protocol.
6
RNA-seq analysis of bovine endometrium and allantochorion
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The RNA-seq was performed for in total 12 samples: Six endometrium and six allantochorion (three samples in each group, RFM and control in each tissue). According to the protocol, 400 ng of total RNA was used to cDNA libraries preparation using TruSeq RNA Library Prep Kit v2 (Illumina, San Diego, CA, USA). The libraries were ligated with different indexes to pool together during sequencing and exclude the possible lane effect. The libraries were amplified in 15 cycles of PCR, quality and quantity were checked using TapeStation 2200 (with Agilent D1000 ScreenTape; Agilent, Santa Clara, CA, USA) and Qubit (with Qubit dsDNA BR Assay Kit; Invitrogen, ThermoFisher Scientific, Whaltam, MA, USA). Next, the libraries were sequenced in 150 pair-end cycles on HiSeq 3000/4000 according to the protocol (Illumina, San Diego, CA, USA).
7
RNA Extraction and cDNA Synthesis
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1x ice-cold Hank’s balanced salt solution (HBSS, #14175129, Gibco) was added to the human fibroblasts immediately after culture medium was discarded. Fibroblasts were scraped off flasks by scraper, followed by centrifugation at 300 × g, 4°C for 5 min. Cells were washed twice with ice-cold 1x HBSS. Collected cell pellet was then immediately processed to RNA extraction using RNeasy mini kit (#74106, Qiagen), according to the manufacturer’s instructions. Total RNA was dissolved in DNase/RNase-free water, and extracted RNA was kept on ice onwards from now. RNA concentration and quality were measured by a Nanodrop spectrophotometer (IsoGen Life Science). RNA integrity was checked by capillary electrophoresis using Agilent D1000 ScreenTape on the TapeStation 2200 (Agilent Technologies). Subsequently, 1 μg of total RNA was reverse transcribed to cDNA by using an iScript kit (#1708891, Bio-Rad) on a Mastercycler (Eppendorf), with 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C. one cDNA synthesis reaction with an RNA sample without reverse transcriptase (the minRT reaction) was included as a negative control for further real-time RT-PCR analysis. cDNA was stored at −20°C until use.
8
RNA-seq and small-RNA-seq library preparation
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Barcoded RNA-seq and small-RNA-seq libraries were generated according to the manufacturer's protocols using the Ion Total RNA-Seq Kit v2 and the Ion Xpress RNA-Seq Barcoding Kit (Thermo Fisher Scientific), except for the small RNA libraries in which both cDNA and amplified cDNA were subjected to a single step purification using a higher ethanol volume to increase the selected fragment sizes (up to 200 nt). The size distribution and yield of the barcoded libraries were assessed using the 2200 TapeStation System with Agilent D1000 ScreenTapes (Agilent Technologies). Sequencing templates were prepared on an Ion Chef System using the Ion PI Hi-Q Chef Kit (Thermo Fisher Scientific). Sequencing was performed on an Ion Proton System using Ion PI Chips v3 (Thermo Fisher Scientific) according to the instructions of the manufacturer.
9
Small RNA Sequencing Library Preparation
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Small RNA sequencing libraries were prepared using NEXTFLEX Small RNA-Seq Kit v3 (Bio Scientific Corp., TX, United States) using the manufacturer’s protocol. Briefly, 500 ng of total RNA was used as starting material. 3′ adapters were ligated to the specific 3′OH group of microRNAs followed by ligation of 5′ adapter. Adapter-ligated fragments were reverse transcribed with Moloney murine leukemia virus (M-MuLV) reverse transcriptase by priming with reverse transcription primers. Complementary DNA (cDNA) thus formed was enriched and indexed by PCR (15 cycles). Libraries were size selected using the NEXTflex magnetic bead purification system and finally reconstituted in nuclease-free water. The sequencing libraries were quantified by the Qubit fluorometer (Thermo Fisher Scientific, United States), and its fragment size distribution was analyzed on Tape Station using Agilent D1000 Screen Tapes (Agilent Technologies, United States). The libraries were sequenced on Illumina NextSeq 500 sequencer (Illumina, Inc., United States) for 75-bp single read chemistry following manufacture’s guidelines.
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