GSM6735312: A393; Homo sapiens; RNA-Seq
Eight mL fresh blood were collected from FXS male individuals (N=29) and age-matched typically developing (TD) males (N=13)  in a K2EDTA Tube and the leukocytes collected on a LeukoLOCKTM filter prior to RNA extraction using a LeukoLOCK Fractionation & Stabilization Kit, Ambion #1933 as per the manufacturer's instructions. Briefly, the blood samples were passed through LeukoLOCKTM filters that were then rinsed with 3 mL phosphate buffered saline (PBS) followed by 3 mL of RNAlater®. The filters were capped and stored in -80 deg C. To extract RNA, the filters were thawed at room temperature for 5 mins and then the remaining few drops of RNAlater were removed. The filter was flushed with 4 ml of TRIzol™ LS Reagent (ThermoFisher Scientific #10296028) and the lysate collected in a 15 ml tube. 800 μl bromo-3-chloro-propane (BCP) (Sigma #B9673) was added to each tube and vortexed vigorously for 30 sec. The tube was then incubated at room temp for 5 min and centrifed for 10 min at 40C at ~2,000 x g;  the aqueous phase containing the RNA was recovered. To recover the long RNA fraction, 0.5 volumes of 100% ethanol were added and mixed well. The RNA was then recovered using an RNA clean and concentrator kit (Zymo Research, #11-325 / R1015), DNase treated with TURBO™ DNAse (Invitrogen # AM2238) and the RNA resuspended in RNAse free water and stored at –80°C. Three mg of RNA sample was used for directional mRNA library preparation using polyA enrichment (Novogene Co) and the libraries sequenced on the NovaSeq platform to generate paired end, 150bp reads at a sequencing depth of 60 million reads per sample