Biomek fxp liquid handler

RNA was extracted from 200 µl of sera using the Agencourt RNAdvance blood extraction kit (Beckman Coulter) following manufacturer’s instructions and involving a DNase treatment step at 37°C for 15 minutes as previously described.¹ (link) Extracted RNA was converted to cDNA using Superscript III (Invitrogen) and random hexamers. The cDNA was converted to double-stranded DNA using the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs). Library preparation was performed using the KAPA LTP Library Preparation Kit from Kapa Biosystems, and the process was automated using the Biomek FXP liquid handler from Beckman Coulter. NEBNext Multiplex Oligos were utilized for indexing and amplification.¹¹ The amplified libraries were quantified using a Qubit 3.0 fluorimeter from Invitrogen and a 4200 TapeStation from Agilent. The libraries were then pooled in equal molar amounts and sequenced using the NextSeq 500/550 High Output v2.5 300 cycle kit, employing a paired-end read configuration with a length of 2 × 150 bp. In order to lower the chance of cross-contamination, a stringent one-way sequencing system with separated rooms for each stages of the NGS sequencing process was followed.

Samples were extracted using the Qiagen RNeasy mini kit (#74104 rev. 10/19) following the manufacturer’s protocol for samples <5e6 cells. Samples were eluted in 50ul RNase-free water. RNA quality and quantity were estimated using Agilent Bioanalyzer OR Caliper GX. To monitor sample and process consistency, 1 μl of the 1:50 diluted synthetic RNA designed by External RNA Controls Consortium (ERCC) (4456740, ThermoFisher) was added. Whole transcriptome sequencing (total RNAseq) data was generated using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Globin kit (20020612, Illumina Inc.) cDNA was prepared following rRNA and Globin mRNA depletion, and paired-end libraries were prepared on Beckman BioMek FXp liquid handlers. For this, cDNA was A-tailed followed by ligation of the TruSeq UD Indexes (Cat # 20022370) and amplified for 15 PCR cycles following manufacturer’s recommendation. AMPure XP beads (A63882, Beckman Coulter) were used for library purification. Libraries were quantified using a Fragment Analyzer (Agilent Technologies, Inc) electrophoresis system and pooled in equimolar ratios. This pool was quantified using qPCR to determine loading concentration for sequencing. Sequencing was performed on the NovaSeq 6000 instrument using the S4 reagent kit (300 cycles) to generate 2×150bp paired end reads.