title: RNA-seq of human blood samples from MiBLEND study on the effect of fruit and vegetable blends on gene expression
design: RNA-seq of human blood samples from MiBLEND study on the effect of fruit and vegetable blends on gene expression
library_name: Sample 119_p
library_construction_protocol: Whole blood samples were collected from participants using standard venipuncture techniques into EDTA-coated tubes during specific test days. On the collection day, participants arrived at the research facility after fasting overnight. Immediately after blood collection, the samples were transferred into freezer-safe Eppendorf tubes containing RNAlater solution (Ambion, Austin, TX, USA) in a 2:1 ratio (RNAlater to blood volume). This step was taken to stabilize the RNA in the samples. The tubes were then stored at -80°C until further processing. The samples were collected both at baseline and post-intervention phases, ensuring uniform handling across all time points. RNA extraction from the blood samples was performed using the Directzol RNA microprep kit (ZY-R2052, Zymo Research), following the manufacturer's protocol. Before extraction, the blood samples were thawed on ice, and the RNAlater solution was removed by centrifugation at 5000× g for 1 minute. After the supernatant was discarded, 1xPBS (phosphate-buffered saline) was added to wash the pellet, followed by centrifugation to remove remaining impurities. QIAzol lysis reagent (QIAGEN) was then added at a 3:1 ratio, and the mixture was vortexed to ensure thorough lysis. Chloroform was added for phase separation, and the samples were centrifuged at 1200× g for 15 minutes at 4°C. The RNA-containing aqueous phase was transferred to a new tube, mixed with ethanol, and processed using Directzol spin columns. The extracted RNA was eluted in RNase-free water, and RNA quality and concentration were measured using a Qubit Fluorometer (Life Technologies) and a Bioanalyzer (Agilent Technologies). Only samples with RIN (RNA Integrity Number) greater than 7 were used for further analysis. RNA libraries were prepared using the NextFlex Rapid Directional RNA kit 2.0 with ribo-depletion (Perkin Elmer, Waltham, MA, USA). Approximately 100 ng of total RNA was used for each library preparation, with ribo-depletion carried out to remove rRNA. The libraries were constructed following the manufacturer's guidelines and were normalized according to RNA concentration and library size. The prepared libraries were checked for quality and fragment size distribution using a Bioanalyzer (Agilent Technologies).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14563:Sample 119_p