HLA genotyping from the amplicon assay NGSgo-AmpX was used as the benchmark reference. NGSgo-AmpX consists of dedicated primer sets for the amplification of individual HLA genes, enabling the amplification of the following HLA genes: Class I: HLA-A, HLA-B, and HLAC-C; and Class II: HLA-DRB1 and HLA-DQB1 (GenDx, Utrecht, Netherlands). Three capture-based assays include 1) Agilent SureSelect Human All Exon V5+UTR kits and paired-end sequencing (150PE) strategies were carried out using standard Illumina protocols on an Illumina HiSeq X10 system (WES for short). Each sample met the average depth over 100X and capture on-target ratio >50%. 2) IDT xGen® Exome Research Panel kits and paired-end sequencing (150PE) strategies were carried out using standard Illumina protocols on an Illumina HiSeq X10 system (Bofuri for short). Each sample met the average depth over 100X and capture on-target ratio >60% (10 samples were not available). 3) 3DMed Inc. in-house designed and developed HLA specific probes and paired-end sequencing (150PE) was carried out using standard Illumina protocols on an Illumina HiSeq X10 system (Internal for short). Each sample met the average depth over 100X and capture on-target ratio >60%. The raw fastq files from Miseq sequencing were subsequently processed and validated by the vendor independently, and used as the benchmarked result for HLA typing.
Hiseq x10 system
Manufactured by Illumina
Sourced in United States, China
The HiSeq X10 system is a high-throughput DNA sequencing platform manufactured by Illumina. It is designed to enable rapid and efficient genome sequencing at a large scale. The system utilizes Illumina's proprietary sequencing-by-synthesis technology to generate DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Trueseq stranded mrna platform, by Illumina (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nebnext ultra 2 directional rna seq kit, by New England Biolabs (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Poly a rna selection kit, by Lexogen (2 mentions)
Mirneasy micro kit, by Qiagen (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq x10 platform, by Illumina (2 mentions)
4200 bioanalyzer, by Agilent Technologies (2 mentions)
Vahts mrna seq v2 library prep kit for, by Illumina (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Edta vacuum blood collection tubes, by BD (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
Magattract kit, by Qiagen (1 mentions)
Chromium instrument, by 10x Genomics (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Trueprep dna library prep kit v2, by Illumina (1 mentions)
32 protocols using hiseq x10 system
1
Comprehensive HLA Genotyping Evaluation
2
Exome Sequencing from Peripheral Blood DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood was obtained from the participants using disposable EDTA vacuum blood collection tubes (BD Biosciences), and DNA was extracted using the QIAamp DNA Blood Midi Kit (Qiagen GmbH). Qualified genomic DNA was randomly fragmented by Covaris technology (200 and 300 bp) and adapters were ligated to both ends of the resulting fragments. The DNA was amplified by ligation-mediated PCR, purified and hybridized to an exome array for enrichment. Non-hybridized fragments were then removed by washing. The exomes were captured using a MedE006 kit (MyGenostics) and each qualified captured library was loaded onto an Illumina HiSeq X-10 system (Illumina Inc.). High-throughput sequencing of the captured library was performed to ensure that the samples met the desired average sequencing coverage. Sequencing-derived raw image files were processed using the Illumina HiSeq X-10 system with default parameters and the sequencing data were generated as paired-end reads, which were defined as ‘raw data’ and stored in FASTQ format. The detailed process is depicted in Fig. 1 .
3
RNA m6A Immunoprecipitation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The qualified RNA (more than 100 μg and RIN > 7.0) was retained for sequencing in the next step. Then the poly (A) RNA solution was fragmented at 94 °C for 5 min using the thermocycler. Subsequently, the pre-equilibrated m6A-Dynabeads was added into the fragmented RNA at room temperature while rotating (tail-over-head) at 7 rotations per minute for one hour. After washing of m6A-Dynabeads, elution of m6A-positive RNA and extraction and cleanup step of the RIP, a RIP library was constructed using 100 ng of RNA (100 ng of input and 100 ng of post m6A-IP positive fraction) utilizing the Illumina TrueSeq Stranded mRNA platform. Finally, Illumina HiSeq X10 system (OE Biotech Co., Ltd., Shanghai, China) was performed to conduct the 2×150 bp paired-end sequencing. The raw data in this work was deposited at the GENE EXPRESSION OMNIBUS (GEO) database (accession number: PRJNA879097).
4
Whole Genome Sequencing of BXD Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anesthetized mice with 5% isoflurane from 40 BXD RI strains and their B6 and D2 parental stains were euthanized by cervical dislocation. Spleen tissue was collected immediately, flash-frozen with liquid nitrogen, and stored at −80 °C for subsequent analysis. The DNA extraction, library preparation, and WGS were carried out by HudsonAlpha (Huntsville, AL, USA). Briefly, genomic DNA was isolated from 50 to 80 mg of spleen tissue using the Qiagen MagAttract Kit (Qiagen, Germantown, MD, USA). The Chromium Gel Bead and Library Kit (v2 HT Kit, revision A; 10X Genomics, Pleasanton, CA, USA) and the Chromium instrument (10X Genomics) were used to prepare libraries for sequencing; barcoded libraries were then sequenced on the Illumina HiSeq X10 system (Illumina, Inc., San Diego, CA, USA).
5
Low-pass Whole-Genome Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For LC-WGS, libraries were prepared using the Kapa Hyper Prep kit (Roche, CA, United States) with custom adapters (IDT, CA, United States) starting with 50 to 1000 ng of DNA input (median, 471 ng), which was used for low-pass WGS. The 22 Libraries were pooled and sequenced using the 150-base paired-end runs over 1× lane on a HiSeq X10 system (Illumina, CA, United States). Segment copy numbers were derived using a customized workflow ultrasensitive chromosomal aneuploidy detector (UCAD). If the median absolute deviation of the copy ratio (log ratio) between the adjacent bins of the whole-genome was greater than 0.38, indicating poor-quality sequence data, the sample was excluded.
Reads were mapped to the EBV reference genome (gi|82503188). Matches with no more than 1 mismatch were counted as EBV reads. The same approach was applied for H. pylori (gi|261838873). Samples with more than 4 EBV reads were marked as EBV-positive tumor samples. Samples with more than 4 H. pylori reads were marked as H. pylori-positive tumor samples.
Reads were mapped to the EBV reference genome (gi|82503188). Matches with no more than 1 mismatch were counted as EBV reads. The same approach was applied for H. pylori (gi|261838873). Samples with more than 4 EBV reads were marked as EBV-positive tumor samples. Samples with more than 4 H. pylori reads were marked as H. pylori-positive tumor samples.
6
Transcriptome Analysis of Renal Cortex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transcriptome analysis was conducted by Seqhealth Technology Co., LTD (Wuhan, China). Total RNA from the renal cortex of control Pacs-2fl/fl mice and control PT-Pacs-2−/− mice was extracted using RNAiso Plus (TaKaRa, Japan) according to the manufacturer’s instructions. The RNA concentration was determined by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and the integrity was tested by 1.0% agarose gel electrophoresis. 5 μg total RNAs were used for mRNA sequencing library preparation. After the library quality inspection is qualified, ~ 300 bp products were sequenced in HiSeq X10 system (Illumina, San Diego, CA, USA). For the RNA-seq data analysis, the data was aligned to the mouse reference genome to obtain comprehensive transcript information and differentially expressed genes (DEGs) as described previously (Ma et al. 2021 (link)).
7
Single Worm RNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single worm RNA sequencing used a protocol modified from single-cell RNA sequencing for mouse cells62 (link). A single young adult animal was transferred into 2 μl lysis buffer and lysed by grinding. In all, 1 μl of each oligo-dT primer (10 μM) and dNTP (10 mM) was added into the PCR tube and heated at 72°C for 3 mins then cooled for 2 min. 6 μl reverse transcription mixture (100 U SuperScript II reverse transcriptase (Takara), superscript II first-strand buffer, 1 U RNAase inhibitor (Vazyme),10 M betaine (Sigma), 6 mM MgCl2 (Ambion), and 100 μM TSO primer) was then added directly and incubated using thermal cycle: 90 min at 42 °C, 15 min at 72 °C and hold at 4 °C. cDNA samples were amplified with 10 μl KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and 12.5 μl 10 μM IS PCR primers. The purified cDNA was fragmented by TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Inc) Hiseq X 10 system sequencing. Primers are listed in Supplementary Table 3 .
8
Single-cell sequencing of PBMCs and kidneys
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Several different sequencing methods were used for the samples of PBMCs and kidneys. For PBMCs, each sample was diluted to a final concentration of 700–1200 cells µl−1 and loaded onto a Chromium Single Cell Controller (10X Genomics, San Francisco, USA). The libraries for scRNA-seq were constructed using a Chromium Next GEM Single Cell 3´ GEM, Library and Gel Bead Kit v3.1 (10X Genomics) and then sequenced using an Illumina NovaSeq 6000 system. For each kidney sample, the single-cell suspension was adjusted to a concentration of approximately 300 cells µl−1. A GEXSCOPE Single Cell RNA Library Kit (Singleron Biotechnologies) was then used to construct a single-cell RNA-seq library for kidney samples. The libraries were then sequenced with an Illumina HiSeq X 10 system. Each sample of PBMCs and kidney tissue was processed independently.
9
RNA-seq Analysis of Human Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were homogenated in lysis buffer and RNA was extracted as user manual (AllPrep DNA/RNA Mini Kit, Cat.no 80204, Qiagen). RNA samples were then processed to library construction. RNA and library DNA quantity were evaluated with Qubit 3.0 and suitable kit. RNA and library size distributions were measured with Aglient Bioanalyzer 2100 or QIAxcel system. High-throughput sequencing was performed with Illumina Hi-seq X10 system. RNA-seq reads were aligned to hg19 human genome and transcript annotation (GENCODE). Gene raw read counts were calculated via feature counts in R package Subread. Differentially expressed genes (DEGs) analysis was perform using DESeq2 [77 (link)]. DESeq2 was run with the formula “~ group + gender”, where group has levels “Group I” and “Group II” and “gender” has levels “male” and “female”. Genes with adjusted p-value less than 0.05 defined as DEGs.
10
Abiotic Stress Response Profiling in Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNA-Seq was performed on samples from 10-day-old whole seedlings and roots. Whole seedlings were treated with 150 mM NaCl for 1, 2, and 4 h or 300 mM mannitol for 1, 2, and 4 h each. Subsequently, the samples were harvested, and either complete (whole seedlings) or fractionated (roots only) samples were used for RNA isolation. After total RNA isolation, 2 μg of each sample was mixed and used for mRNA-Seq library preparation, which was performed by E-biogen (https://www.e-biogen.com , accessed on 13 September 2022), as previously described [51 (link)]. High-throughput paired-end 100 bp sequencing was performed using a HiSeq X10 system (Illumina, Inc., San Diego, CA, USA). Two biological replicates of each sample were used for RNA-Seq.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!