Rosette leaf tissues from four- to five-week-old Col-0 and representative T2 MBD2–ZF108 transgenic lines that display the early flowering phenotype were collected for BS-PCR at the FWA promoter regions. DNA was extracted using the DNeasy Plant Mini kit (Qiagen). Around 2 μg of DNA was used for DNA bisulfate conversion with the EpiTect Bisulfate kit (QIAGEN). The converted DNA served as a template for amplification. PCR was performed at three different regions spanning the promoter and the 5′ transcribed regions of FWA, including region 1 (chr4: 13038143–13038272), region 2 (chr4: 13038356–13038499) and region 3 (chr4: 13038568–13038695). The PCR reactions used Pfu Turbo Cx (Agilent), dNTP (Takara Bio) and the primers designed for the above-mentioned FWA regions. PCR products from the same sample were pooled and cleaned using AMPure beads (Beckman Coulter). The purified PCR products were prepared for the libraries using the Kapa DNA Hyper Kit (Roche) with indexes from TruSeq DNA UD indexes for Illumina (Illumina). Finally, the libraries were sequenced on an Illumina iSeq 100.
Kapa dna hyper kit
Manufactured by Roche
The Kapa DNA Hyper Kit is a molecular biology tool that enables efficient and high-yield DNA amplification. It provides a reliable and consistent method for generating large quantities of DNA from small starting samples. The kit includes all the necessary components for the DNA amplification process, allowing researchers to perform their experiments with ease and confidence.
Automatically generated - may contain errors
Lab products found in correlation
Pfu turbo cx, by Agilent Technologies (6 mentions)
Iseq 100, by Illumina (4 mentions)
Ampure bead, by Beckman Coulter (3 mentions)
Epitect bisulfite kit, by Qiagen (3 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Epitect bisulfite conversion kit, by Qiagen (2 mentions)
Truseq dna adapters, by Illumina (2 mentions)
Nextera dna library kit, by Illumina (2 mentions)
Epitect bisulfate kit, by Qiagen (1 mentions)
Truseq dna ud indexes, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Ovation ultralow v2 kit, by Tecan (1 mentions)
Ovation ultralow methyl seq kit, by Tecan (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Hiseq 6000, by Illumina (1 mentions)
10 protocols using kapa dna hyper kit
1
BS-PCR Analysis of FWA Promoter
2
Whole-genome Bisulfite Sequencing of Arabidopsis FWA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rosette leaves of about one-month-old plants were collected and subject to DNA extraction with CTAB method followed by bisulfite DNA conversion using the EpiTect Bisulfite kit (QIAGEN) kit. Three regions of the FWA gene were amplified from the converted DNA with Pfu Turbo Cx (Agilent): Region 1 (chr4: 13038143-13038272), Region 2 (chr4: 13038356- 13038499) and Region3 (chr4: 13038568-13038695). Primers used are listed in Supplementary Table 4 . Libraries were prepared with the purified PCR product by the Kapa DNA Hyper Kit (Roche) together with TruSeq DNA UD indexes for Illumina (Illumina) and were sequenced on Illumina iSeq 100 or HiSeq 4000 instruments.
BS-PCR-seq data was analyzed as previously described43 (link). Briefly, raw reads were aligned to both strands of the TAIR10 reference genome with BSMAP (v.2.90)66 (link) allowing up to 2 mismatches and 1 best hit. After quality filtering, the methylation level of cytosines was calculated as the ratio of C/(C+T), and customized R scripts were used to plot methylation data over the FWA region 1-3.
BS-PCR-seq data was analyzed as previously described43 (link). Briefly, raw reads were aligned to both strands of the TAIR10 reference genome with BSMAP (v.2.90)66 (link) allowing up to 2 mismatches and 1 best hit. After quality filtering, the methylation level of cytosines was calculated as the ratio of C/(C+T), and customized R scripts were used to plot methylation data over the FWA region 1-3.
3
Bisulfite Sequencing of FWA Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rosette leaves of about one-month-old plants were collected and subject to DNA extraction with CTAB method followed by bisulfite DNA conversion using the EpiTect Bisulfite kit (QIAGEN) kit. Three regions of the FWA gene were amplified from the converted DNA with Pfu Turbo Cx (Agilent): Region 1 (chr4: 13038143-13038272), Region 2 (chr4: 13038356- 13038499) and Region3 (chr4: 13038568-13038695). Primers used are listed in Supplementary Data 4 . Libraries were prepared with the purified PCR product by the Kapa DNA Hyper Kit (Roche) together with TruSeq DNA UD indexes for Illumina (Illumina) and were sequenced on Illumina iSeq 100 or HiSeq 4000 instruments.
BS-PCR-seq data was analyzed by aligning the raw reads to both strands of the TAIR10 reference genome with BSMAP (v.2.90)67 (link) allowing up to 2 mismatches and 1 best hit. After quality filtering, the methylation level of cytosines was calculated as the ratio of C/(C + T), and customized R scripts were used to plot methylation data over the FWA region 1-3.
BS-PCR-seq data was analyzed by aligning the raw reads to both strands of the TAIR10 reference genome with BSMAP (v.2.90)67 (link) allowing up to 2 mismatches and 1 best hit. After quality filtering, the methylation level of cytosines was calculated as the ratio of C/(C + T), and customized R scripts were used to plot methylation data over the FWA region 1-3.
4
Bisulfite Sequencing of FWA Promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The leaf tissue from 4- to 5-week-old Col-0 wild type, fwa and the representative T2 ZF lines showing early flowering phenotype were collected to perform BS-PCR at FWA promoter regions. Cetyltrimethyl ammonium bromide-based method was used to extract DNA, and the EpiTect Bisulfite kit (QIAGEN) was used for DNA conversion. The converted DNA was used as a template to amplify three different regions over promoter and 5′ transcribed regions of FWA, including region 1 (chr4: 13038143-13038272), region 2 (chr4: 13038356- 13038499) and region 3 (chr4: 13038568-13038695). Pfu Turbo Cx (Agilent), dNTP (Takara Bio) and the primers designed for the above-mentioned FWA regions (Supplementary Table 8 ) were used to perform PCR reactions. Three different PCR products from three regions of each sample were pooled and purified with AMPure beads (Beckman Coulter). The purified PCR products were used to construct libraries by the Kapa DNA Hyper Kit (Roche) together with TruSeq DNA UD indexes for Illumina (Illumina), and the libraries were sequenced on Illumina iSeq 100.
5
Yeast Genomic DNA Sequencing
6
Bisulfite Sequencing of FWA Promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BS-PCR-seq was performed as previously described34 (link). Briefly, leaf tissues from adult plants of representative T2 lines containing the ZF-SssI transgene were collected and DNA was extracted following a CTAB-based method. Bisulfite conversion was done using the Epitect Bisulfite Conversion kit (QIAGEN). The following regions of FWA were analyzed: Region 1 (chr4: 13038143-13038272), Region 2 (chr4: 13038356-13038499), and Region3 (chr4: 13038568-13038695); which cover fragments of the promoter and 5′ transcribed region of FWA. Pfu Turbo Cx (Agilent) was used to amplify bisulfite-treated DNA using primers containing Illumina adaptors. The primers used are listed in Supplementary Data 10 .
For each sample, individual PCR products from each of the three FWA regions were pooled and purified using AMPure beads (Beckman Coulter) before making the libraries. Libraries were made from purified PCR products using a TruSeq Nano DNA Library Prep kit for Neoprep automated library preparation machine (Illumina), a Kapa DNA hyper kit (Kapa Biosystems) with Illumina TruSeq DNA adapters, or an Ovation Ultralow V2 kit (NuGEN).
For each sample, individual PCR products from each of the three FWA regions were pooled and purified using AMPure beads (Beckman Coulter) before making the libraries. Libraries were made from purified PCR products using a TruSeq Nano DNA Library Prep kit for Neoprep automated library preparation machine (Illumina), a Kapa DNA hyper kit (Kapa Biosystems) with Illumina TruSeq DNA adapters, or an Ovation Ultralow V2 kit (NuGEN).
7
Genome-wide Bisulfite Sequencing for Plant DNA Methylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For T2 and T3 WGBS, DNA from inflorescences of adult plants was extracted following a CTAB-based method. Hundred nanograms of DNA were sheared to 200 bp with a Covaris S2 (Covaris) and used for library preparation using the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer’s instructions. For T4 and T5 WGBS, DNA from inflorescences of adult plants was extracted using the DNeasy Plant Mini Kit (QIAGEN). Two hundred fifty nanograms of DNA were sheared to 200 bp with a Covaris S2 (Covaris) and used for library preparation using the Epitect Bisulfite Conversion kit (QIAGEN) and the Kapa DNA hyper kit (Kapa Biosystems) with Illumina TruSeq DNA adapters following the manufacturer’s instructions.
8
Bisulfite Sequencing of DNA Methylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rosette leaves from representative T2 and T3 were collected. DNA was extracted with a CTAB-based method followed by RNase A (Invitrogen) treatment to remove RNA. Bisulfite conversion was performed with Epitech Bisulfite Conversion Kit (Qiagen) following the manufacturer’s instructions. Three regions were amplified for the methylation analysis: chr4: 13038143-13038272, chr4: 13038356-13038499, and chr4: 13038568-13038695. The amplification was performed with Pfu Turbo Cx (Agilent). Libraries were made from purified PCR products using Kapa DNA hyper kit (Kapa Biosystems) with Illumina TruSeq DNA adapters. Libraries were sequenced on Illumina HiSeq 6000. BS PCR primers are listed in Supplementary Table 1 .
9
Illumina Sequencing of Drosophila Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed 2×150 bp Illumina sequencing for most of the strains that did not have publicly available short read data available. Illumina libraries were prepared from the same gDNA extractions as the Nanopore library for most samples, with some exceptions as described in Supplementary file 1 . The libraries were prepared in either of two manners. For the majority of samples, sequencing libraries were prepared with a modified version of the Nextera DNA Library Kit (Illumina, San Diego, CA) protocol (Baym et al., 2015 (link)) and sequencing was performed by Admera Health on NextSeq 4000 or HiSeq 4000 machines. Alternatively, Illumina libraries were prepared with the KAPA Hyper DNA kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol and sequenced at the UNC sequencing core on a HiSeq 4000 machine. In either case, all samples on a lane were uniquely dual indexed. Illumina sequencing was not performed for D. equinoxialis, D. funebris, D. subpulchrella, D. tropicalis, Le. varia, Z. lachaisei, Z. taronus, and the unidentified São Tomé mushroom feeder due to material unavailability (line extinction/culling). Details for each sample, including accession numbers for any public data used in this work, are provided in Supplementary file 1 .
10
Illumina Sequencing of Drosophila Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed 2Â150 bp Illumina sequencing for most of the strains that did not have publicly available short read data available. Illumina libraries were prepared from the same gDNA extractions as the Nanopore library for most samples, with some exceptions as described in Supplementary file 1. The libraries were prepared in either of two manners. For the majority of samples, sequencing libraries were prepared with a modified version of the Nextera DNA Library Kit (Illumina, San Diego, CA) protocol (Baym et al., 2015) (link) and sequencing was performed by Admera Health on NextSeq 4000 or HiSeq 4000 machines. Alternatively, Illumina libraries were prepared with the KAPA Hyper DNA kit (Roche, Basel, Switzerland) according to the manufacturer's protocol and sequenced at the UNC sequencing core on a HiSeq 4000 machine. In either case, all samples on a lane were uniquely dual indexed. Illumina sequencing was not performed for D. equinoxialis, D. funebris, D. subpulchrella, D. tropicalis, Le. varia, Z. lachaisei, Z. taronus, and the unidentified Sa ˜o Tome ´mushroom feeder due to material unavailability (line extinction/culling). Details for each sample, including accession numbers for any public data used in this work, are provided in Supplementary file 1.
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!