GSM2367168: six RNA samples of young donors were pooled 4Y_S19; Homo sapiens; miRNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated according to the method of Neitzel (Neitzel 1986) with modifications. Briefly, 4 ml of full blood were centrifuged at 2500×g for 20 min at 4°C. The bottom layer of approximately 2 ml, containing all types of blood cells, was diluted in 4 ml of phosphate-buffered saline (PBS) and the resulting suspension was overlayed onto 3 ml of lymphocyte separation medium LSM 1077 (PAA Laboratories GmbH). The samples were centrifuged at 600×g for 20 min at 19°C. The layer of PBMCs was collected and washed twice with PBS. Total RNA was extracted from PBMCs with TRIzol Reagent (Invitrogen) according to the manufacturer protocol. PBMCs were lysed in TRIZOL® Reagent by repetitive pipetting. The homogenized samples were incubated for 5 min at 15-30°C. Next, 0.2 ml of chloroform per 1 ml of TRIZOL® Reagent was added, samples were capped, shaken vigorously by hand for 15 s, incubated at 15-30°C for 2-3 min and centrifuged at no more than 12,000 × g for 15 min at 2-8°C. Total RNA was solely in 1 among 3 separated phases - the aqueous phase which then was transferred to a fresh tube and added 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL® Reagent.  Afterwards, samples were incubated at 15-30°C for 10 min and centrifuged at no more than 12,000 × g for 10 min at 2-8°C. Supernatant was discarded and RNA pellet was washed by 1 ml of 75% ethanol per 1 ml of TRIZOL® Reagent. The sample was mixed by vortexing and centrifuged at no more than 7,500 × g for 5 min at 2-8°C. Obtained RNA pellet was briefly drained and subsequently dissolved in 20 ul of RNase-free water. Equal amounts of total RNA from each study participant (250 ng per person)  were pooled, 4 pools of 6 RNA samples each per age group, and subjected to miRnome analysis. The quality and integrity of RNA was verified on Agilent 2100 Bioanalyzer (Agilent, CA, USA). The RIN of used RNA > 8. Small RNA library was prepared with TruSeq Small RNA Library Preparation kit (Illumina Inc., CA, USA). Total RNA was used for direct ligation of adapters to miRNAs. 3’ adapter in Illumina kit is modified, thus it binds only to 3’ hydroxyl group of small RNAs. Next ligated miRNAs were reverse transcribed, amplified with primers containing unique indexes and pooled. The fraction of small RNAs library was extracted using Pippin HT (Sage Science, MA USA) and sequenced on the Illumina NextSeq 500 platform. Small RNA fraction was sequenced and analyzed as described previously [Swierniak et al.,. 2013].