Total RNA was isolated from snap frozen oral epithelial cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The isolated RNA samples were checked for quality control using the RNA 6000 Nano Chip kit in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries for RNA-seq were prepared from total RNA (300 ng per sample) using the Kapa Hyper Prep Kits with RiboErase (Kapa Biosystems, Wilmington, DE, USA). The workflow consisted of rRNA depletion, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation, and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Nextseq500 for a single-end read for 75 cycles. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v2.17 program. To rule out any potential bias, library construction and data acquisition for samples from different groups (vapers, smokers, and controls) were done in the same run, not in different batches, and in a “blind” fashion. Detailed descriptions of data processing and analysis are provided in Supplementary Data . The RNA-seq data will be deposited in the Gene Expression Omnibus database at NCBI (htttp://www.ncbi.nlm.nih.gov/geo/ ), and accession number will be provided as soon as it becomes available.
Bcl2fastq2 v 2.17 program
Manufactured by Illumina
Bcl2fastq2 v 2.17 is a program that converts base call (BCL) files generated by Illumina sequencing instruments into FASTQ format files. The program's core function is to demultiplex and convert Illumina sequencing data into the standard FASTQ format, which can then be used for downstream bioinformatics analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 3000, by Illumina (15 mentions)
Agilent tapestation, by Agilent Technologies (5 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Hiseq 3000 4000, by Illumina (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Rneasy ffpe kit, by Qiagen (3 mentions)
Rna 6000 nano chip kit, by Agilent Technologies (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Nanodrop 8000, by Agilent Technologies (2 mentions)
Blood rna kit, by Qiagen (2 mentions)
Globinclear human kit, by Thermo Fisher Scientific (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Kapa hyper prep kits with riboerase, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Library construction kit, by 10x Genomics (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Stranded rna seq kit, by Roche (1 mentions)
29 protocols using bcl2fastq2 v 2.17 program
1
RNA Extraction and RNA-seq from Oral Epithelial Cells
2
Single-Cell RNA-seq Library Preparation
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After the identities of the cDNA amplification products were confirmed, the sequencing library was constructed using a Library Construction KIT (10× Genomics). First, the cDNA was chemically knocked-out. The cDNA fragment was then cut into 200~300 bp fragments, the cDNA fragments were segmented, and their terminals were repaired and added. The cDNA fragments were then screened. The P7 adapter was connected and introduced into the sample index using PCR amplification. Finally, a sequence library was obtained. Sequencing was performed on an Illumina Hiseq 3000/4000 (Illumina, San Diego, CA, USA) with a 150 bp pair-end run by Quick Biology (Pasadena, CA, USA). A data quality check was performed using the SAV (Illumina). Demultiplexing was performed using the Bcl2fastq2 v 2.17 program (Illumina).
3
RNA-Seq Analysis of Endothelial Cell Subsets
4
RNA Extraction and Sequencing from FFPE Tissues
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To isolate RNA, 10-µM-thick paraffin slices were trimmed from each FFPE tissue block using a microtome. RNA was extracted from FFPE slices using QIAGEN RNeasy FFPE Kit following the manufacturer's protocol. RNA 6000 Nano or Qubit RNA Assay kits were used to measure RNA concentration. The RNA integrity number (RIN) was measured using the Agilent 2100 Bio-Analyzer. For depletion of ribosomal RNA and library construction, the KAPA RNA Hyper with rRNA erase kit (HMR only) was used. Different adaptors were used for multiplexing samples in one sequencing run. Library concentrations and quality were measured using the Qubit ds DNA HS Assay kit (Life Technologies) and Agilent TapeStation. RNA sequencing was done at the Department of Pathology and Laboratory Medicine, University of California Los Angeles, using Illumina HiSeq 3000 equipment for single-end sequencing, 50-bp read length, for ∼30 million (mln) raw reads per sample. A data quality check was done on Illumina SAV. Demultiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program. Sequencing data were deposited in the NCBI SRA under accession ID PRJNA562149. Summary statistics of RNA sequencing (number of unmapped, uniquely mapped, and multimapped reads as well as average exon coverage) is depicted in Supplemental Table S1 .
5
Transcriptome Analysis of BEAS-2B Cells
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Total RNA from BEAS-2B cells was extracted using a RNeasy Plus Mini kit (Qiagen). Libraries were then prepared by Quick Biology (Pasadena, USA) using a KAPA Stranded RNA-Seq Kit. The workflow included mRNA enrichment, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation, and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000/4000 for a pair end 150 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. F-box protein mRNA levels in the glucose free treatment group were normalized to the high glucose media group.
6
Transcriptome Profiling via Illumina RNA-Seq
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Libraries for RNA-Seq were prepared with KAPA Stranded mRNA-Seq Kit. The workflow consists of mRNA enrichment, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina HiSeq 3000 for 1 × 50 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program.
7
RNA-Seq Library Preparation and Analysis
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Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit on RNA isolated from livers of chow diet feeding mice with AAV-GFP or AAV-Cre transduction. The data were sequenced on Illumina HiSeq 3000 for a pair-end 150 bp read run. Data quality check were done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. RNA-seq reads were aligned with TopHatv2.0.2 to the mouse genome, version mm9 25. Transcripts were assessed and quantities were determined by Cufflinks v2.0.2, using a GTF file based on Ensembl mouse NCBI37. Comparison expression levels were made using Fragments Per Kilobase of transcript per Million (FPKM) values using Cuffdiff from the Cufflinks package 26.
8
Stranded RNA-Seq Protocol with Multiplexing
9
RNA Extraction from FFPE Tissues
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To isolate RNA, 10 µM - thick paraffin slices were trimmed from each FFPE tissue block using microtome. RNA was extracted from FFPE slices using QIAGEN RNeasy FFPE Kit following the manufacturer’s protocol. RNA 6000 Nano or Qubit RNA Assay kits were used to measure RNA concentration. RNA Integrity Number (RIN) was measured using Agilent 2100 bio-Analyzer. For depletion of ribosomal RNA and library construction, KAPA RNA Hyper with rRNA erase kit (HMR only) was used. Different adaptors were used for multiplexing samples in one sequencing run. Library concentrations and quality were measured using Qubit ds DNA HS Assay kit (Life Technologies) and Agilent Tapestation (Agilent). RNA sequencing was done at Department of Pathology and Laboratory Medicine, University of California Los Angeles, using Illumina HiSeq 3000 equipment for single-end sequencing, 50 bp read length, for approximately 30 million (mln) raw reads per sample). Data quality check was done on Illumina SAV. De-multiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. Sequencing data were deposited in NCBI Sequencing Read Archive (SRA) under accession ID PRJNA663280.
10
RNA Sequencing Protocol for Transcriptomics
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RNA sequencing was done according to Suntsova et al. (2019 (link)) and Sorokin et al. (2020d (link)) at the Department of Pathology and Laboratory Medicine, University of California Los Angeles. For depletion of ribosomal RNA and library construction, KAPA RNA Hyper with rRNA erase kit (HMR only) was used. Different adaptors were used for multiplexing samples in one sequencing run. Library concentrations and quality were measured using Qubit ds DNA HS Assay kit (Life Technologies) and Agilent Tapestation (Agilent). RNA sequencing was done using Illumina HiSeq 3000 equipment for single-end sequencing, 50 bp read length, for approximately 30 million (mln) raw reads per sample. Data quality check was done on Illumina SAV. De-multiplexing was performed with Illumina Bcl2fastq2 v 2.17 program.
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