Small rna sample prep kit

Total RNA of IMF-preadipocytes and IMF-adipocytes were prepared using mirVana™ miRNA Isolation Kit (Austin TX, US). After quality checking, ~2 μg total RNA for each sample was used to construct the small RNA library according to the protocol (Illumina Small RNA Sample Prep Kit). A total of 4 libraries (IMF-preadipocytes and IMF-adipocytes, each with 2 replicates) were sequenced with Illumina Genome Analyzer (Illumina, San Diego, CA, USA). Meanwhile, cDNA was also prepared using PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian, China) and stored at −20 °C for the following wet-lab experiment.

Roots from mock and V. dahliae-treated cotton were used for RNA library construction and deep sequencing analyses according to [37 (link)]. Total RNA was isolated by using the RNA reagent (Invitrogen, Carlsbad, CA, USA). The quantity and quality of the isolated total RNA were assessed using a NanoDrop OneC Spectrophotometer. For transcriptome sequencing, the enriched mRNAs were purified from the total RNA with magnetic beads attached to oligo (dT) and the strand-specific libraries were sequenced on Illumina Hiseq2500 platform at Novogene (Beijing, China) with pair-end strategy (2 × 150 bp). Moreover, small RNA libraries were constructed using the Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequencing was performed on Hiseq platform at Novogene (Beijing, China) with single end strategy (50 bp).

RNA from protonemal tissus of the WT and mutant lines were grown for 7 days then subjected to 24 hours of ABA treatment, dehydration, and rehydration (see Fig. 2A). These samples were extracted separately, following the protocol in1 (link). The protonemal tissues from the two mutant lines at corresponding stages were mixed before RNA extraction. Total RNA was isolated using the Plant RNA Isolation Mini kit (Agilent Technologies, Inc., DE, USA) following the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed by ThermoScript™ RT-PCR System (Invitrogen Co., CA, USA) with oligo-dT(20) primer following the manufacturer’s instruction. PCR was carried out using 1 μL of cDNA with GoTaq^® Green Master mix (Promega Co., WI, USA). Small-RNA libraries were prepared for Illumina sequencing following the manufacturer’s instructions40 (link). miRNeasy Mini Kit (Qiagen) was used for RNA extraction, with on-column DNase I digestion, followed by sequencing on the Illumina GAIIx platform using Small RNA Sample Prep Kit (Illumina).

Low-molecular-weight RNAs were isolated from the adult testes of w¹¹¹⁸ and tsn¹¹²⁰/tsn⁰⁶¹⁴ mutant males (approximately 200 pairs for each genotype) using a mirVana miRNA isolation kit (Life Technologies). Small RNAs ranging in size between 16 and 29 nucleotides (nt) (below 2S rRNA) were gel-purified, and small RNA libraries were prepared using a small RNA sample prep kit (Illumina) according to the alternative v1.5 protocol. The clones were sequenced using Genome Analyzer II. Only sequences perfectly matching the Drosophila melanogaster release 5 genome (excluding Uextra) were analyzed. As TSN is known to regulate miRNAs [17 (link)], here we used the total reads instead of the reads of miRNAs to normalize the two libraries. After removal of miRNAs and fragments of long cellular RNAs such as tRNAs and rRNAs, we mapped 18–32-nt unique piRNAs to a subset of known piRNA clusters and to the complete collection of Drosophila melanogaster transposable elements (Repbase) [37 (link)].

Total RNA was isolated from the root samples using an RNA kit according to the manufacturer’s instructions (EASYspin for plant RNA, Beijing, China). The seven RNA samples, including the samples from the six inoculation time points and the mock-inoculated, were used for RNA-seq. RNA samples were digested with DNase I (Qiagen, Hilden, Germany), and the quality and quantity were determined using a NanoDrop 2000 (Thermo Scientific, NH, USA) and an Agilent 2100 (Agilent, Santa Clara, CA, USA) instrument. RNA of each sample was purified using oligo(dT)-attached magnetic beads from an mRNA-Seq Sample Prep Kit (Illumina, San Diego, CA, USA). The purified mRNA was used for preparing a non-directional Illumina RNA-seq library using a Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The library’s quality and quantification were analysed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and an ABI Step One Plus Real-Time PCR System (ABI, CA, USA). Each library was applied to an Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) for single-end sequencing by the Beijing Genomics Institute (Shenzhen, China). Raw sequences were transformed into clean reads after data processing, leaving 49 nt tags.