Tracheal cells (epithelial, immune and mesenchymal) were lysed in 350 ul of Trizol, and RNA was extracted by phenol:chloroform method, and collected over RNAeasy column (Qiagen, Germantown, MD). The RNA concentration and integrity was determined on the NanoDrop ND-1000 (Thermo Fisher Scientific). 1000ng RNA from each sample was used as a template for preparing Illumina compatible libraries using the TruSeq RNA Library Prep Kit v2 (Illumina). Library sizes were checked using D5000 high sensitivity tape on the TapesSation 2200 (Agilent), and pooled libraries concentration were determined by Qubit 3.0 Fluorometer (Thermo Fisher Scientific). A library input of 1.8 pM with 1% PhiX (Illumina) spike-in was sequenced using the NextSeq 500 instrument with the NextSeq500/550 High Output v2.5 Kit (Illumina) and generated paired-end 76bp reads. Samples from all four animals (two dams and two infants) were processed and analyzed together.
Nextseq 500 550 high output v2.5 kit
Manufactured by Illumina
Sourced in United States
The Illumina NextSeq 500/550 High Output v2.5 Kit is a sequencing reagent kit designed for the Illumina NextSeq 500 and NextSeq 550 sequencing systems. The kit enables high-throughput DNA and RNA sequencing for a variety of applications, including whole-genome sequencing, targeted resequencing, transcriptome analysis, and more. The kit includes the necessary reagents, flow cells, and other consumables required to perform sequencing runs on the Illumina NextSeq instruments.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (7 mentions)
Nextseq 500, by Illumina (6 mentions)
Nextseq 500 system, by Illumina (4 mentions)
Nextseq, by Illumina (4 mentions)
Truseq standard mrna lt sample prep kit, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Sh800s cell sorter, by Sony (2 mentions)
Kapa rna hyperprep kit with ribose erase, by Roche (2 mentions)
Nextseq 500 sequencer, by Illumina (2 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rnaeasy column, by Qiagen (1 mentions)
Truseq rna library prep kit v2, by Illumina (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Nextseq 550 sequencer, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Small rnaseq library prep kit, by Illumina (1 mentions)
30 protocols using nextseq 500 550 high output v2.5 kit
1
Tracheal Cell Transcriptomic Analysis
2
Dual Size-Selected Wolbachia Genome Sequencing
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150 ng of DNA was fragmented on a Covaris M220 sonicator 1 n 15 µL tube with parameters optimized for a maximum of fragments sized about 400 bp. Genome libraries were prepared using the Roche KAPA Hyper Prep Kit, KAPA UDI adapters and 50 nanograms of fragmented DNA according to the manufacturer’s manual for dual size selection. Amplification of libraries was carried out in nine cycles. The quality and molarity of the resulting libraries were determined using a Bioanalyzer BA2100. After normalization, barcoded libraries were pooled and sequenced on an Illumina NextSeq550 sequencer (2 × 150 bp) using the NextSeq® 500/550 High Output v2.5 Kit 300 cycles with the expected output for Wolbahia being 30 million reads for the ovarian sample and on an Illumina MiSeq sequencer (2 × 250 bp) using the MiSeq Reagent Kit v2 (Illumina, San Diego, CA, USA) (500-cycles) for the sample from whole flies.
3
Transcriptome Analysis of Hsf5 Mutant Testes
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The seminiferous tubules from whole testes of male Hsf5 +/- (n = 3) and KO (n = 3) mice at P16 were unraveled and incubated in DMEM containing 10% FBS at 33 and 37 °C for 3 h. Total RNAs were prepared by RNeasy Mini Kit (QUIAGEN, 74104). Quality of total RNA was confirmed by BioAnalyzer 2100 (RIn > 9). Library DNAs were prepared according to the Illumina TruSeq protocol using TruSeq. Standard mRNA LT Sample Prep Kit (Illumina) and sequenced by Illumina NextSeq 500 (Illumina) using Nextseq 500/550 High Output v2.5 Kit (Illumina) to obtain single end 75 nt reads.
4
Transcriptome Profiling of Rec8-GFP Testis
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GFP-positive cells from Rec8-3xFLAG-HA-p2A-GFP knock-in male testis were sorted using SH800S cell sorter (SONY). Total RNAs were prepared by Trizol (ThermoFisher) and the quality of total RNA was confirmed by BioAnalyzer 2100 (RIN > 8) (Agilent). Library DNAs were prepared according to the Illumina Truseq protocol using Truseq Standard mRNA LT Sample Prep Kit (Illumina) and sequenced by Illumina NextSeq 500 (Illumina) using Nextseq 500/550 High Output v2.5 Kit (Illumina) to obtain single-end 75 nt reads.
Resulting reads were aligned to the mouse genome UCSC mm10 using STAR ver.2.6.0a after trimmed to remove adapter sequence and low-quality ends using Trim Galore! v0.5.0 (cutadapt v1.16). Gene expression level measured as RPKM was determined by Cuffdiff v 2.2.1. Differential expression analysis using TPM was done by RSEM v1.3.1. GTF file was derived from UCSC mm10. Principal component analysis and GO-term analysis were performed using DAVID Bioinformatics Resources 6.847 (link).
Resulting reads were aligned to the mouse genome UCSC mm10 using STAR ver.2.6.0a after trimmed to remove adapter sequence and low-quality ends using Trim Galore! v0.5.0 (cutadapt v1.16). Gene expression level measured as RPKM was determined by Cuffdiff v 2.2.1. Differential expression analysis using TPM was done by RSEM v1.3.1. GTF file was derived from UCSC mm10. Principal component analysis and GO-term analysis were performed using DAVID Bioinformatics Resources 6.847 (link).
5
RNA Extraction and Quantification Protocol
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TSCs were passaged once off of irMEFs as above onto a single well of a 6-well plate. ESCs were grown on a single well of a 6-well plate. Both were grown to ≥75% confluency prior to RNA harvest using 1mL TRIzol, followed by the addition of 200μL chloroform, which were vortexed and subsequently spun at max speed for 5 min at 4°C for phase separation. The aqueous layer was collected and combined with 1 volume of 100% isopropanol and 5μL linear acrylamide. Precipitation was achieved at −80°C for 1 h, followed by a max speed spin for 30 min at 4°C and one wash of the RNA pellet with ice-cold 80% ethanol. The pellet was then resuspended in 100μL H2O and quantified via Qubit (Invitrogen, cat #: Q32855).
For RT-qPCR assays inFigures 5A and S4E , 1μg of RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit, and qPCR was performed using iTaq Universal SYBR Green Supermix and custom primers (Table S5 ).
RNA-seq libraries were prepared from 1μg of total RNA using KAPA RNA HyperPrep Kit with Ribose Erase (Kapa Biosystems, cat #: KR1351) according to the manufacturer instructions. Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit on a NextSeq 500 System.
For RT-qPCR assays in
RNA-seq libraries were prepared from 1μg of total RNA using KAPA RNA HyperPrep Kit with Ribose Erase (Kapa Biosystems, cat #: KR1351) according to the manufacturer instructions. Single-end, 75-bp sequencing was performed using an Illumina NextSeq 500/550 High Output v2.5 kit on a NextSeq 500 System.
6
Small RNA-Seq Library Preparation
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Aliquots of 100 ng of miRNAs, derived from the above-mentioned procedure, were used for the preparation of miRNA libraries. Sequencing-ready cDNA libraries using the Small RNASeq Library Prep kit for Illumina (Lexogen, Austria) were prepared according to the manufacturer’s instructions. Multiplexing indices were introduced during the PCR amplification step to distinguish each sample after the pooling phase, allowing multiplexing of up to 96 libraries [17 (link)]. The library product was cleaned and concentrated with a magnetic bead-based purification protocol [18 ]. Lastly, the sequencing step was performed with NGS technologies using Illumina platform NextSeq 500 and the NextSeq 500/550 High Output v2.5 kit (150 cycles).
7
Illumina Adapter PCR Library Preparation
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For adapter PCR, 8 µL of each sample was mixed with 1× Phusion Master Mix (Thermo Scientific #F-531), 0.5 µM Illumina adapter, index primer (corresponding to each sample), and nuclease-free water in a total volume of 20 µL. PCR program: 98 °C 2 min; 18 repeats of: 98 °C 20 s, 65 °C 30 s, 72 °C 30 s; 72 °C 5 min; 10 °C. PCR product purification was performed using Agencourt® AMPure XP beads according to the manufacturer’s recommendation (1.8 ratio). Positive controls contained pure barcode oligos (no scFv) and negative control (water). Quality control of purified PCR products was done using Bioanalyzer and Agilent High Sensitivity DNA kit. Five microliters of each sample was pooled, diluted, and prepared for sequencing on a NextSeq 500/550 High Output v2.5 kit (Illumina) on a NextSeq 500 sequencer (Illumina).
8
Transcriptomic analysis of spermatocytes in Hsf5 KO mice
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Spermatocytes at zygotene/pachytene from WT and Hsf5 KO male testis were sorted using SH800S cell sorter (SONY) using DyeCycle Violet (DCV) stain (Thermo Fisher)44 . Total RNAs were prepared by Trizol (Thermo Fisher) and quality of total RNA was confirmed by BioAnalyzer 2100 (RIn > 8) (Agilent). cDNA was synthesized by SMART-Seq mRNA kit (Takara, 634772). Library DNAs were prepared according to the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096) and sequenced by Illumina NextSeq 500 (Illumina) using Nextseq 500/550 High Output v2.5 Kit (Illumina) to obtain single end 75 nt reads.
The resulting reads were aligned to the mouse genome UCSC mm10 using STAR ver.2.7.8a after trimmed to remove adapter sequence and low-quality ends using Trim Galore! v0.6.6 (cutadapt v2.5). Differential expression analysis using TPM was done by RSEM v1.3.3, and DESeq2 (v1.36.0). GTF file was derived from UCSC mm10. Principal component analysis and GO-term analysis were performed using DAVID Bioinformatics Resources 6.875 (link). Sequencing data are available at DDBJ Sequence Read Archive under the accession DRA017058.
The resulting reads were aligned to the mouse genome UCSC mm10 using STAR ver.2.7.8a after trimmed to remove adapter sequence and low-quality ends using Trim Galore! v0.6.6 (cutadapt v2.5). Differential expression analysis using TPM was done by RSEM v1.3.3, and DESeq2 (v1.36.0). GTF file was derived from UCSC mm10. Principal component analysis and GO-term analysis were performed using DAVID Bioinformatics Resources 6.875 (link). Sequencing data are available at DDBJ Sequence Read Archive under the accession DRA017058.
9
cDNA Library Preparation and Sequencing
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The construction of cDNA libraries was performed according to a standard protocol using a NEBNext Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs, UK) for small RNA fraction, NEBNext Ultra II Directional RNA library preparation kit for Illumina (New England Biolabs, Hitchin, UK) and NEBNext mRNA Magnetic Isolation Module (New England Biolabs, UK) for poly(A)+ RNA fraction. For the prepared sequencing libraries, fragment size distribution was analysed using a Bioanalyzer 2100 instrument (Agilent, USA) with an Agilent High Sensitivity DNA Kit (Agilent, USA) and quantification by Qubit DNA HS Assay Kit (Thermo Fisher Scientific, USA) with Qubit 2 fluorometer (Thermo Fisher Scientific, USA). Fragment size ranges between 100 bp to 200 bp and between 250 bp to 700 bp were observed for small RNA and poly(A)+ RNA libraries respectively. Libraries were sequenced on Illumina NextSeq 1500 instrument in 100-base-pair-single-end mode (NextSeq 500/550 High Output v2.5 Kit (Illumina, USA)). The construction of cDNA libraries and massive parallel sequencing were conducted at the Institute of Fundamental Medicine and Biology, Kazan Federal University (Kazan, Russia).
10
Microvesicle RNA Sequencing Protocol
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RNA from the EV cohort and the negative control (RNA extraction from culture medium) were subjected to library preparation. MicroRNA libraries were created with the QIAseq miRNA library kit (QIAGEN, Hilden, Germany). This kit was chosen over the Illumina library preparation kit, as its input requirement was lower and could accommodate the low RNA yields from erythrocyte‐derived EVs. Libraries were pooled and sequenced in single‐end fashion on a NextSeq 500 flow cell (NextSeq 500/550 High Output v2.5 kit , 150 cycles; Illumina, San Diego CA, USA).
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