Fresh leaves and whole flowers of a P. chrysochlora (Primulaceae) plant growing in Tengchong, Yunnan Province, China (25 21′05.74′N, 98 08′18.90′E, alt. 1810 m), were sampled and immediately stored in RNAlater solution (Takara Biotechnology Co., Ltd.) to preserve the RNA. Total RNA was subsequently extracted using a modified hexadecyltrimethylammonium bromide (CTAB) method. Quantified total RNA (concentration ≥100 ng/µL; rRNA ratio ≥1.5) was delivered to Macrogen, where cDNA sequencing was performed with the Roche GS FLX Titanium platform. Raw data were filtered for adapters, low-quality reads, or short reads below 40 bp. For P. chrysochlora, 428,716 cleaned reads with average length of 368 bp (range: 40–1044 bp) were obtained and deposited in the Sequence Reads Archive (SRA) database under accession number SRX1037980.
Rnalater solution
Manufactured by Takara Bio
Sourced in Japan
RNAlater solution is a stabilization reagent designed to rapidly permeate tissues and cells, and protect and stabilize cellular RNA. It is formulated to inhibit RNase activity and preserve the in vivo expression profile of RNA transcripts, allowing for the collection, storage, and transportation of samples prior to RNA extraction and analysis.
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5 protocols using rnalater solution
1
Transcriptome profiling of Primula chrysochlora
2
Colon Adenoma Quantification Protocol
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Mice were killed with an overdose of isoflurane, weighted, and necropsied. The colon of mice was then removed and cut open, and the mucosal surface was photographed in high resolution for grossly visible polypoid adenoma counts. One-centimeter colon samples were collected from a standard area of the proximal part of descending colon for gene expression and ELISA analyses. Samples for gene expression assay were immediately immersed in an RNA-later solution (Takara Bio Inc, Shiga, Japan) and stored at − 80°C until further processing. The remaining colon was fixed in 10% neutral-buffered formalin for histologic and immunohistochemical analyses.
3
Transcriptome Sequencing of Tibetan Amphipods
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Gammarus lacustris individuals were collected from the Tibetan plateau at the altitude of ~4,300 m (94°48′53″E, 28°41′24″N). To prevent degradation of DNA, entire bodies were stored immediately in 100% ethanol after anesthesia in MS-222, and the ethanol was changed twice before extraction of genomic DNA. For full-length and next-generation transcriptome sequencing, whole individuals were immediately immersed in RNAlater solution (Takara, Tokyo, Japan) after collection, and then were frozen in liquid nitrogen until used for RNA extraction. Meanwhile, G. pisinnus individuals for next-generation transcriptome sequencing were collected from a nearby plain in Shanxi Province of China at the altitude of ~510 m (109°7′6″E, 34°5′57″N). Entire shrimps were immediately immersed in RNAlater solution after collection, and then were frozen in liquid nitrogen for storage.
4
Transcriptome and Chloroplast Genome Sequencing of Iris Species
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Plants were harvested from type localities whenever possible in China (Supplementary Table S1 ). Then, we collected the leaves of I. yunguiensis, I. shangrilaensis, I. taiwanensis, I. sinensis, and I. hypsophila, washed them with distilled water, fixed them in RNAlater solution (Takara, Dalian, China) immediately, and stored them in a −80°C freezer for DNA and RNA extraction. DNA quality and DNA concentration were measured on a NanoDrop 2000 (Supplementary Table S2 ).
We sequenced the transcriptomes and chloroplast genomes of the six individuals. Total genomic DNA was extracted using an extract Plant DNA kit (TIANGEN, China), while total RNA was isolated using the RNAiso Plus kit (TaKaRa, Dalian, China). Afterward, a paired-end library with an insert size of 350 bp was constructed using the Truseq Nano DNA HT Sample Prep Kit (Illumina, United States), and the RNA-sequencing library was generated using the VAHTS mRNA-seq v2 Library Prep Kit for Illumina® (Vazyme, NR601).
We sequenced the transcriptomes and chloroplast genomes of the six individuals. Total genomic DNA was extracted using an extract Plant DNA kit (TIANGEN, China), while total RNA was isolated using the RNAiso Plus kit (TaKaRa, Dalian, China). Afterward, a paired-end library with an insert size of 350 bp was constructed using the Truseq Nano DNA HT Sample Prep Kit (Illumina, United States), and the RNA-sequencing library was generated using the VAHTS mRNA-seq v2 Library Prep Kit for Illumina® (Vazyme, NR601).
5
Postmortem Tissue Sampling Protocols
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Rats were purchased from the Department of Animal Science Laboratory of Fudan University. In total, 18 male Sprague-Dawley rats (body weight 220 ± 20 g) were randomly assigned to three groups (mechanical asphyxia, craniocerebral injury and hemorrhagic shock) to collect the anterior region of the brain and the cardiac muscle of the apex cordis. All samples were placed in RNA Later solution (Takara, Japan) immediately after collection. The animal experiments described in the study were performed in accordance with the principles for the Care and Use of Laboratory Animals and were approved by the Science and Ethics Committee of Fudan University. In the mechanical asphyxia death group, a nylon rope (diameter 3 mm) was used to create a sliding loop that was fixed to the top of a bar, and the rats were placed in the loop, causing suffocation death based on their own gravity. In the craniocerebral injury death group, a 10-g weight fell freely from a 20-cm height within the vertical catheter and hit the dura. In the hemorrhagic shock death group, the bilateral carotid artery was isolated and cut to induce death via shock.
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