For bulk RNA sequencing of PBMC samples from the independent Singapore cohort (see above), we performed reverse transcription and amplification using a standard protocol83 (link). Briefly, we synthesized cDNA from 2 ng of purified total RNA using modified oligo(dT) primers, and cDNAs were further amplified. The quantity and integrity of cDNA were assessed using the DNA High Sensitivity Reagent Kit (Perkin Elmer: LabChip GX). Subsequently, pooled cDNA libraries were prepared (250 pg of cDNA per sample, Illumina Nextera XT kit, Cat no. FC-131-1096) with dual indices for de-multiplexing. The libraries were quantified using qPCR (Kapa Biosystems) to ascertain the loading concentration. Samples were subjected to an indexed PE sequencing run of 2 × 151 cycles on an Illumina HiSeq 4000. We mapped paired-end reads to human genome build GRCh38 using the STAR aligner84 and counted reads mapped to genes using featureCounts85 and GENCODE v31 gene annotations86 (link). We quantified gene expression as log2-transformed reads per exonic kilobasepair per million mapped reads (log2RPKM) using the edgeR Bioconductor package87 . Gene expression estimates for SOCS3 were then analyzed for association with disease severity (D.K. et al.49 , unpublished observations).
Dna high sensitivity reagent kit
Manufactured by PerkinElmer
Sourced in United States
The DNA High Sensitivity Reagent Kit is a laboratory tool designed for the detection and quantification of DNA samples. It provides a sensitive and accurate method for measuring DNA concentrations, enabling researchers to obtain reliable data for their DNA-based experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt kit, by Illumina (10 mentions)
Hiseq 2000, by Illumina (6 mentions)
Ercc rna spike in control, by Thermo Fisher Scientific (6 mentions)
Agilent bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq 4000 system, by Illumina (3 mentions)
Hiseq 4000, by Illumina (2 mentions)
Rneasy micro, by Qiagen (2 mentions)
Perkin elmer labchip gx system, by PerkinElmer (2 mentions)
Hiseq 2000 system, by Illumina (2 mentions)
Bsa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyser, by Agilent Technologies (1 mentions)
Advantage 2 pcr kit, by Takara Bio (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Smarter ultra low rna kit, by Takara Bio (1 mentions)
Dna extended range labchip, by PerkinElmer (1 mentions)
Quant it picogreen dsdna reagent, by PerkinElmer (1 mentions)
Bioanalyser, by Agilent Technologies (1 mentions)
Kapa hyper prep, by Roche (1 mentions)
13 protocols using dna high sensitivity reagent kit
1
Bulk RNA-seq of PBMC Samples
2
RNA Isolation, Sequencing, and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from tissue followed by a Qiagen RNeasy Micro clean-up procedure (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNAs were analyzed on Perkin Elmer Labchip GX system (Perkin Elmer, Waltham, MA, USA) for quality assessment with RNA Quality Score ≥ 7.9. cDNA libraries were prepared using 2 ng of total RNA and 1 μl of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Fisher Scientific, Waltham, MA, USA) using SMARTSeq v2 protocol [19 (link)] except for the following modifications: 1. Use of 20 μM TSO, 2. Use of 250 pg of cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip. All samples were subjected to an indexed PE sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000.
Reads were mapped to MM10 with STAR v2.2.3 and gencode M9 annotation. Gene counts were determined with feature counts, differential gene expression with edgeR in R v 3.1.2 [20 (link)]. Data analysis was performed in pipeline pilot (www.accelrys.com ) and plots generated in Spotfire (www.tibco.com ).
Reads were mapped to MM10 with STAR v2.2.3 and gencode M9 annotation. Gene counts were determined with feature counts, differential gene expression with edgeR in R v 3.1.2 [20 (link)]. Data analysis was performed in pipeline pilot (
3
Single-cell RNA-seq using SMARTSeq v2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell cDNA libraries were using the SMARTSeq v2 protocol79 (link) with the following modifications: (1) 1 mg/ml BSA lysis buffer (Ambion Thermo Fisher Scientific, Waltham, MA, USA); (2) use of 250 pg of cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an indexed paired-end sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA) (192 samples/lane). Pair-endraw reads were aligned to human reference genome using RSEM version 1.3.0. Human reference genome version 25 released by Gencode was used (https://www.gencodegenes.org/human/release_25.html ). Transcript Per Million read(TPM) values were calculated using RSEM version 1.3.0 and used for downstream analysis.
4
Comprehensive RNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All human RNAs were analyzed on the Agilent Bioanalyzer (Agilent, Santa Clara, CA, USA) or the Perkin Elmer Labchip GX system (Perkin Elmer, Waltham, MA, USA) for quality assessment with RNA Integrity Number (RIN) or RNA Quality score range from 6.8–9.7 and median of 9.0. cDNA libraries were prepared using 2 ng of total RNA and 1 µL of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Fisher Scientific, Waltham, MA, USA) using SMARTSeq v2 protocol [16 (link)], except for the following modifications: (1) Use of 20 µM TSO, and (2) use of 250 pg of cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip GX system (Perkin Elmer, Waltham, MA, USA). Sixteen samples were subjected to an indexed PE sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000 (16 samples/lane), and 65 samples to an indexed PE sequencing run of 2 ×151 cycles on an Illumina HiSeq 4000 (30 samples/lane).
5
Single-cell RNA-seq library preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
cDNA libraries were prepared using 2 ng of total RNA and 1μL of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Scientific) using SMARTSeq v2 protocol [21 (link)] with modifications listed in Table S5. Length distribution of the cDNA libraries was monitored using DNA High Sensitivity Reagent Kit on Perkin Elmer Labchip (Perkin Elmer). All samples were subjected to an indexed PE sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000 (16 samples/lane).
6
Bulk RNA-seq from Human Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was extracted following the double-extraction protocol: RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction (TRIzol, Thermo Fisher Scientific, Waltham, MA, USA) followed by a Qiagen RNeasy Micro clean-up procedure (Qiagen, Hilden, Germany). All human RNAs were analyzed on the Agilent Bioanalyzer for quality assessment with RNA integrity number (RIN) range from 6.2 to 9.6 and median RIN 8.9 (Agilent, Santa Clara, CA, USA). cDNA libraries were prepared using 1 ng of the total RNA and 0.5 µl of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion Thermo Fisher Scientific, Waltham, MA, USA) using SMARTSeq v2 protocol79 (link), except for the following modifications: (1) use of 20 µM TSO, (2) use of 250 pg of cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an indexed PE sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000 (16 samples/lane). The paired-end reads were mapped to Human GRCh38 reference genome using the STAR alignment tool. The number of reads mapped to each gene was counted using feature Counts (part of Subread package) and GENCODE gene annotation version V25.
7
RNA-Seq profiling of primary keratinocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from primary cultured normal human epidermal keratinocytes (NHEK) following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (TRIzol, Thermo Fisher Scientific, Waltham, MA, USA) followed by a Qiagen RNeasy Micro clean-up procedure (Qiagen, Hilden, Germany). RNA was analyzed on Agilent Bioanalyser for quality assessment with RNA Integrity Number (RIN) range from 9.6 to 9.8 and median of RIN 9.8. cDNA libraries were prepared using 2 ng of total RNA using the SMARTSeq v2 protocol (31 (link)) with the following modifications: 1. Addition of 20 µM TSO; 2. Use of 200 pg cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an indexed paired-end sequencing run of 2x151 cycles on an Illumina HiSeq 4000 system (Illumina) (31 samples/lane).
8
Single-Cell RNA-Seq of Myeloid Progenitor Cells
9
RNA-seq Library Prep with Globin Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified RNA was analyzed on Bioanalyser (Agilent) for quality assessment. RNA samples with RNA Integrity Number (RIN) of more than 6 were selected for the study (RIN ranging from 6.3 to 9.1 and with a median of 7.5) (Supplemental Table 1 ). cDNA libraries were prepared by Smart-Seq v2 [21 (link)], using a modification of the GlobinLock (GL) method [22 (link)] to block transcription of globin mRNA. Human “DNA 3 long A” and “DNA 3 long B” oligonucleotides (0.6 pmol each) were added to 2 ng of total blood RNA in 2.3 μL, denatured at 95 °C for 30 s, incubated at 60 °C for 10 min for GL oligo hybridization, and held at 42 °C for the loading of the reverse transcriptase (RT) mixture. RT and subsequent steps were according to Smart-Seq v2 with the following modifications: (1) addition of 20 μM template switching oligos (TSO) and (2) use of 200 pg cDNA with 1/5 reaction of Nextera XT Kit (Illumina). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the LabChip (Perkin Elmer). All samples were subjected to an indexed paired-end sequencing run of 2 × 151 cycles on a HiSeq 4000 system (19 samples/lane; Illumina).
10
KAPA Hyper Prep Library Production Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One μg of 3' RACE amplicons were diluted to 20 ng/µl in elution buffer and used as input in a modified KAPA Hyper Prep library production protocol (Kapa Biosystems, MA, USA) to generate libraries for Illumina sequencing. Since amplicon sizes ranged from about 100 -800 bp, the fragmentation step in the protocol was skipped. End repair and addition of a single adenine (A) extension was completed as instructed, followed by ligation to indexed adapters overnight at 4°C. A 0.8X AxyPrep (Thermo Scientific, MA, USA) post-ligation bead cleanup was carried out with final elution in 20 μl of TE buffer. Ligated products were enriched with PCR under the following conditions: initial denaturation at 98°C for 45 s; 4 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 1 min. PCR products were subsequently cleaned with AxyPrep beads at 1X concentration and eluted in 30 μl of TE buffer.
Final library products were assessed for quality on LabChip GX using the DNA High Sensitivity Reagent Kit (Perkin Elmer, MA, USA), diluted to 2 nM concentration and pooled. This was followed by denaturation with sodium hydroxide and further dilution to 6 pM in hybridization buffer for loading into a MiSeq system. Sequencing was set up in accordance with Illumina's recommended protocol for an indexed 100-cycle paired-end run.
Final library products were assessed for quality on LabChip GX using the DNA High Sensitivity Reagent Kit (Perkin Elmer, MA, USA), diluted to 2 nM concentration and pooled. This was followed by denaturation with sodium hydroxide and further dilution to 6 pM in hybridization buffer for loading into a MiSeq system. Sequencing was set up in accordance with Illumina's recommended protocol for an indexed 100-cycle paired-end run.
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!