Qiaquick pcr extraction kit

Total RNA was extracted from the two replicates of the drought and control plants with TRIzol Reagent (Invitrogen, 15596–026) according to the manufacturer’s instructions. The four RNA samples that were of sufficient quality were used to construct the transcriptome sequence library. The total four RNA from each sample was then pooled to one, using equivalent quantities of each sample. The transcriptome sequencing library was generated using NEBNext Ultra RNA Library Prep Kits for Illumina(NEB, USA) following manufacturer’s instructions. Following the instructions provided by Illumina, mRNA was purified from the pooled, total RNA using polyT oligo-attached magnetic beads (Novogene, China). Fragmentation buffer was added to disrupt the mRNA into short fragments. Reverse transcriptase and random primers were used to synthesise the first strand cDNA from the cleaved mRNA fragments. The second strand cDNA was synthesised using buffer, dNTPs, RNase H, and DNA polymerase I. The double strand cDNA was purified using QIAquick PCR extraction kits (QIAGEN, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors were ligated onto the fragments. The required fragments were purified by AMPure XP beads and enriched by PCR to construct a library for transcriptome sequencing.

RNA was extracted using RNA extraction kits from Huayueyang Biotechnology Co., LTD. (China), following the manufacturer’s instructions. The mRNA was enriched using Oligo-dT beads (Qiagen, USA), and fragmented into short fragments by using fragmentation buffer and reverse transcribed into cDNA using random primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTPs and buffer. Then, the cDNA fragments were purified using Qiaquick PCR Extraction Kits (Qiagen, USA), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected using agarose gel electrophoresis and fragments ranging from 140 to 200 bp were excised for PCR amplification. The amplified fragments were sequenced using Illumina HiSeqTM 4000 at the Gene Denovo Biotechnology Co. (China).

Ten µg of total RNA equally from 5 larvae in each group was used to isolate mRNA using oligo(dT) magnetic beads. The cDNA library of each sample was constructed using NEBNext® mRNA Library Prep Reagent Set (NEB, Ipswich, MA, USA) following the manufacturer’s protocols. Briefly, enriched poly(A) RNA of each sample was fragmented into 200–700 nt pieces with RNA Fragmentation Reagents. The cleaved RNA fragments were transcribed into the first-strand cDNA using random hexamer-primers, followed by second-strand cDNA synthesis. The resulting double-stranded cDNA (dsDNA) was purified with QiaQuick PCR extraction kit (Qiagen, Hilden, Germany) and resolved in EB buffer. The purified dsDNA was treated with T4 DNA Polymerase and T4 Polynucleotide Kinase for end-repairing and dA-tailing. After that, they were ligated to sequencing adaptors with barcode using T4 DNA ligase. Finally, fragments with around 200bp-length were purified with QiaQuick GelPurify Kit (Qiagen, Hilden, Germany), and used as templates for PCR amplification to create the cDNA library. The library was paired-end sequenced using PE90 strategy on Illumina HiSeq™ 2000 (Illumina, San Diego, CA, USA) in the Beijing Genome Institute (Shenzhen, China). The challenged and control libraries were sequenced in one lane then raw-reads were sorted out by barcodes.

Total RNA were isolated from the paired tissue samples of five TNBC patients using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

We used Plant RNA Assistant Kit (Kebaiao, Beijing, China) to extract the total RNA from ground samples, according to the manufacturer’s instructions, and quantified the integrity of the RNA by agarose gel electrophoresis and Agilent 2100 Bioanalyzer. The purity and concentration of the RNA were determined by Nanodrop 2000 (Thermo Specific, USA).
The RNA-Seq samples were used to construct 18 libraries (FER-FNM1, FER-FNM2, FER-FNM3, STE-FNM1, STE-FNM2, and STE-FNM3; each sample had three replicates). After total RNA was extracted, mRNA was enriched by Oligo (dT) beads and then fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTP, and buffer. Then, the cDNA fragments were purified with QiaQuick PCR extraction kit (QIAGEN, Valencia, CA, USA), were end-repaired, had poly (A) added, and were ligated to Illumina sequencing adapters. The ligation products were size-selected by agarose gel electrophoresis, PCR-amplified, and sequenced using Illumina HiSeqTM 4000 (Illumina, San Diego, CA, USA).

The total RNA was extracted from the sorted mIgM⁺ B lymphocytes using Trizol reagent kit (Invitrogen) according to the manufacturer’s protocol. RNA quality was determined on a NanoDrop 8000 spectrophotometer (Thermo Scientific) and checked using RNase free agarose gel electrophoresis. After evaluating the quality of the total RNA, the eukaryotic mRNA was enriched by Oligo (dT) beads (Thermo Fisher). Then, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq™ 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China).

We homogenized each cochlea sample and extracted total RNA using a TRIzol Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. After checking the amount of RNA degradation by RNase-free agarose gel electrophoresis and capillary electrophoresis with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), we performed a poly (A)-capture to remove rRNA; then, mRNA was reverse-transcribed into first-strand cDNA using random primers. Next, we synthesized the second-strand cDNA using DNA polymerase I, RNase H, dNTP, and second-strand buffer. After purification using a QiaQuick PCR Extraction kit (Qiagen, Hilden, Germany), the cDNA fragments were end-repaired, poly(A)-tailed, and ligated to Illumina sequencing adapters. The ligation products were -elected by agarose gel electrophoresis and PCR amplification. Sequencing was carried out on an Illumina HiSeq X Ten (Illumina, San Diego, CA, USA) with 2 × 150-bp paired-end reads. All raw reads were deposited in the NCBI Short Read Archive (SRA) Database under SRA accession PRJNA515764.