Rna 6000 nano chip kit

The cotton short fiber mutant Li₂ was developed as a near-isogenic line (NIL) with the WT upland cotton line DP5690 as described before [6] (link). Growth conditions and fiber sampling were previously described [6] (link). Cotton bolls were harvested at the following time-points during development: day of anthesis (DOA), 1, 3, 5, 8, 12, 16, and 20 days post-anthesis (DPA). Cotton fibers were isolated from developing ovules using a glass bead shearing technique to separate fibers from the ovules [56] (link). Total RNA was isolated from detached fibers using the Sigma Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO) with the optional on column DNase1 digestion according to the manufacturer’s protocol. The concentration of each RNA sample was determined using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). The RNA quality for each sample was determined by RNA integrity number (RIN) using an Agilent Bioanalyzer 2100 and the RNA 6000 Nano Kit Chip (Agilent Technologies Inc., Santa Clara, CA) with 250 ng of total RNA per sample.

RNA was isolated as previously described [19] (link). To separate the fibers from the ovules the samples were shaken vigorously enough to break fibers without damaging the ovules. Isolation of RNA was conducted using the Sigma Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO) with on-column DNaseI digestion and used according to the manufacturer’s instructions. RNA quantity was determined by using a Nanodrop 2000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). RNA integrity number (RIN) was determined for each sample using an Agilent Bioanalyzer 2100 and the RNA 6000 Nano Kit Chip (Agilent Technologies Inc., Santa Clara, CA). Only samples with RIN values of 7.0 or higher were used for expression analysis.

Young leaves were collected from parental cultivars and 550 RILs. DNA was isolated as previously described [48 (link)]. The concentration of DNA samples was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA), whereas quality was estimated on 1.5% agarose gel.
Cotton fibers were isolated from developing ovules using a glass bead shearing technique to separate fibers from the ovules [49 (link)]. Developing fibers from 12 to 16 DPA ovules were manually separated by forceps. Total RNA was isolated from detached fibers using the Sigma Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO) with the optional on column DNase1 digestion according to the manufacturer’s protocol. The concentration of each RNA sample was determined using a NanoDrop 2000. The RNA quality for each sample was determined by RNA integrity number (RIN) using an Agilent Bioanalyzer 2100 and the RNA 6000 Nano Kit Chip (Agilent Technologies Inc., Santa Clara, CA) with 250 ng of total RNA per sample. A detailed description of reverse transcription and qPCR for quantification of mRNA transcripts was previously reported [50 , 51 (link)]. 18S rRNA was used as the endogenous reference gene for relative quantitation of the gene expression data. The relative expression levels were calculated using the 2^−ΔΔCt method [52 (link)]. Sequences of primers are listed in Additional file 5.

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.

The isolation of DNA and RNA was performed using the RNA/DNA/PROTEIN Purification Plus Kit (Norgen Biotek, Canada). Briefly, the brain structures (prefrontal cortex, HIP, and dSTR) from rats that underwent cocaine abstinence with extinction training were homogenized (30 s at 3000 rpm, then 2 × 30 s at 2500 rpm; Bioprep-24 Homogenizer (Aosheng, China)) with ceramic beads and lysis buffer. RNA samples were eluted in nuclease-free water preheated to 60°C and purified from DNA (RNA Clean-Up kit; Syngen, Poland). The quantity and quality of the isolated RNA samples were determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, USA) and agarose gel electrophoresis, and the RNA integrity was checked using chip-based capillary electrophoresis with an RNA 6000 Nano Chip Kit and an Agilent Bioanalyzer (Agilent Technologies, USA).

MagMAX™ for Stabilized Blood tubes RNA Isolation Kit (Ambion by Life Technologies, Carlsbad, CA, USA) was used for RNA extraction. The concentration and purity of isolated total RNA was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity number (RIN) was assessed by microfluidic capillary electrophoresis using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Chip Kit (Agilent Technologies, Santa Clara, CA, USA) RNA samples exhibited RNA integrity numbers (RIN) between 7.8 and 9.6 (mean RIN 8.8), indicating optimal RNA quality for downstream applications. RNA from the 6 donors was pooled based on concentration for RNA-seq and kept separate for other downstream experiments at −80 °C.