title: GSM8162583: whole_blood_donor1; Homo sapiens; OTHER
library_construction_protocol: Blood samples were obtained from three human donors with approval from the ethics committee from the Australian National University (ANU) under ethics protocol no. ETH.1.16.01/ETH.01.15.015. Whole blood was collected at the Centre for Personalised Immunology (ANU) in 8.5ml BD Vacutainer Acid Citrate Dextrose (ACD-A) Blood Collection Tubes. For each blood sample, total RNA was extracted using the PureLink RNA Mini kit (Thermo Fisher Scientific). The manufacturer's instructions were followed and 4ml sample of blood per donor was used. On-column DNase treatment was additionally performed by adding 2 units of DNase I in its recommended buffer to the column-bound sample (New England BioLabs) and incubating at 37oC for 15 minutes. The resultant RNA eluate was additionally purified using AMPure XP SPRI beads (Beckman Coulter Life Sciences) according to the manufacturer's recommendations. Briefly, the eluate RNA was supplemented with 1.2× volumes of the SPRI bead suspension and the resultant mixture incubated at room temperature for 5 minutes with periodic mixing. The SPRI beads were brought down by a brief 2,000×g spin and separated from the solution on a magnetic rack. The supernatant was removed, and the beads were resuspended in 1ml of 80% v/v ethanol, 20% v/v deionized water mixture and further washed by tube flipping. The bead and solution separation procedure and the ethanol washing process were repeated one more time. Any remaining liquid was brought down by a brief spin and removed using a pipette, and the beads were allowed to air-dry while in the magnetic rack for 2 minutes prior to elution. The purified RNA was then eluted in 20ml of deionized water and the RNA content was assessed using absorbance readout via Nanodrop. For each of the whole blood samples, 200-600ng of total RNA was used for each 2× library preparation for each sample (all recommended volumes doubled-up). Libraries were made using the direct RNA sequencing kit SQK-RNA002 (Oxford Nanopore Technologies) following manufacturer's recommendations. The final adaptor-ligated sample was eluted in 20μl (single preparation volume). The flow cell priming was performed as follows​. The flow cells were left at 25 °C for 30 minutes to reach ambient temperature. The flow cells were then inserted into the MinION Mk1B and a quality check was performed to ensure that the pore count was above manufacturer warranty level (800 pores). Prior to the sample loading, the priming solution (Flush Buffer mixed with Flush Tether) was degassed in a vacuum chamber for 5 minutes. A similar approach was repeated when loading the RNA library. Nanopore sequencing was performed using an ONT MinION Mk1B equipped with R9.4.1 flow cells; samples were run for 24 hours. Additionally, for the third sample (whole_blood_donor3_liverun) RISER was run at the same time as the MinKNOW sequencing run. In the RISER code, the flow cell channels were split into two groups to remove the effect of inter-flow cell variability by simultaneously testing two conditions: (1) RISER depleting globin mRNA, and (2) no RISER (control). To split the flow cell channels, the channel numbers divisible by 2 were used for the RISER deplete condition (1) while the remaining channels were used for the control condition (2). RISER was started after the first pore scan finished and was run for the same duration as the sequencing run; 24 hours. The live run was performed on a computer running Ubuntu 20.04 with one NVIDIA GeForce GTX 1650 Ti GPU and python v3.8.10. Nanopore direct RNA sequencing