title: GSM9021959: Patient_F_BENRALIZUMAB_WEEK_24;SR8243; Homo sapiens; RNA-Seq
library_name: GSM9021959
library_construction_protocol: Blood samples were aliquoted into 1 mL per tube and treated with filtered (0.2μM, Cytiva, Cat. No. 10462205) 10X RBC Lysis Buffer Multi-species (eBioscience, Cat. No. 00-4300-54) for optimal lysis of erythrocytes. White blood cells were collected by centrifugation at 500 x g for five minutes at 4°C. The resulting pellets were re- suspended and homogenized in 1 mL of QIAzol Lysis Reagent (Qiagen, Cat. No. 79306) and stored at − 80°C. On the day of RNA extraction, samples were thawed and allowed to return to room temperature. For every milliliter of QIAzol, 0.2 mL of the phase separation reagent chloroform (Fisher Scientific, Cat. No. 10784143) was added. The samples were then homogenized by vigorously shaking to an emulsion. After an incubation time of two minutes at RT, samples were centrifuged at 10.000 x g for fifteen minutes at 4°C to separate the phases. The upper aqueous phase ( ∼ 500 μL) was transferred to a new microcentrifuge tube. In this tube, 500 μL of isopropanol (Fisher Scientific, Cat. No. 10111733) and 2 μL of GlycoBlue (Fisher Scientific-Invitrogen, Cat. No. 10391565), to increase pellet visibility, were added. After an incubation time of ten minutes at RT, samples were centrifuged at 10.000 x g for fifteen minutes at 4°C. Supernatant was then removed and the pellet was washed with 500 μL of 75% cold ethanol (Sigma-Aldrich, Cat. No. 1085430250) to precipitate the RNA. Samples were again centrifuged at 10.000 x g for one minute at 4°C and each pellet was re-suspended in RNase-free water after air drying. RNase-free conditions were maintained at all times. Total RNA extraction was followed by column-based RNA purification with the RNA Clean & Concentrator-5 kit (Zymo Research, Cat. No. R1013). Before clean- up, the DNase digestion procedure was performed using the DNase I Set. DNase I reaction mix (50 μL) was prepared and incubated at RT for fifteen minutes. Following the purification protocol, two volumes of RNA Binding Buffer (100 μL) and an equal volume (150 μL) of 100% ethanol (Sigma-Aldrich, Cat. No. 1085430250) were added to each sample and transferred to a Zymo-Spin IC column. The columns were first centrifuged at 10.000 × g for thirty seconds. This was followed by one wash with RNA Prep Buffer and two washes with RNA Wash Buffer, following the manufacturer's instructions. Final elution of 15 μL each, with centrifugation at 10.000 x g for thirty seconds at RT, was performed with RNase-free water. The purified and concentrated RNA samples were stored at − 80°C. RNase-free conditions were maintained at all times. Sequencing libraries were prepared with the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (Illumina, Cat. No. 20040529), following the manu- facturer's protocol. The input amount of total RNA was 500 ng per sample. The sequencing libraries were quantified by qPCR using the KAPA SYBR FAST qPCR Master Mix and primers targeting the Illumina P5 and P7 flow cell oligo sequences (Roche Diagnostics, Cat. No. 07959389001). Libraries were then pooled in equimolar amounts. The amount of libraries loaded onto the flow cell was 1 nM. The clustered flow cells underwent paired-end sequencing (2 × 150 bp) on a NovaSeq 6000 Sequencing System (Illumina, Cat. No. 20012850), with the NovaSeq S4 Reagent Kit 300 cycles v1.5 kit (Illumina, Cat. No. 20028312), yielding an average of 33 x 106 reads per sample.