4.2. BRB-seq and Data Processing

Multiplexed cDNA libraries were generated using the BRB-seq workflow [12]. First-strand synthesis and barcoding of cDNA was performed by the addition of 5.75 µL DNase-free water, 1 µL of 10 µM oligo-dT primers and 1 µL of dNTP (10 mM) to 6 µL of RNA followed by 5 min incubation at 65 °C. This was followed by reverse transcription by the addition of 2 µL of DTT (Invitrogen), 0.25 µL of SuperScript II enzyme (Invitrogen), 4 µL of SuperScript II 5X Buffer (Invitrogen, Waltham, MA, USA), and 1 µL of 10 µM template-switching oligo (Integrated DNA Technologies, Coralville, IA, USA) in TE buffer (Invitrogen, AM9858). The mix was then incubated at 42 °C for 50 min, followed by inactivation at 70 °C for 15 min in a thermal cycler. To pool and concentrate the samples, the five pools of barcoded cDNA were then purified using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. This was immediately followed by exonuclease treatment by the addition of E. coli Exonuclease I (NEB, #M0293) and incubation at 37 °C for 30 min followed by enzyme inactivation at 80 °C for 20 min, resulting in 20 µL of pooled and exonucleated cDNA. The second-strand cDNA synthesis was performed by the addition of 1 µL of 10 µM LA_oligo (Integrated DNA Technologies), 1 µL of 0.2 mM dNTP, 1 µL of Advantage 2 polymerase mix (Clontech, #639206), 5 µL of Advantage 2 PCR buffer (Clontech, #639206) and 22 µL of DNase-free water, and then PCR amplification (95 °C, 1 min, then 10 cycles of 95 °C for 15 s, 65 °C for 30 s, 68 °C for 6 min.; and finally 72 °C for 10 min). The five cDNA pools were then purified using AMPure XP magnetic beads (Beckman Coulter, #A63881) according to the manufacturer’s instructions. Then, 1 ng of purified cDNA was tagmented (Illumina, Nextera XT DNA library Prep Kit) by adding 10 µL of Nextera ‘Tagment DNA buffer’ and 5 μL of ‘Amplicon Tagment mix’, then incubated at 55 °C for 5 min, and finally the Tn5 transposase was dissociated by the addition of 5 µL of ‘NT Buffer’ followed by incubation at room temperature for 5 min. Then, the indexing of tagmented DNA was performed by the addition of 1 µL of 10 µM P5 BRB oligo (Integrated DNA Technologies), 1 µL of 10 µM Nextera N70X oligos, 8 µL of RNase/DNase-free water and 15 µL of Nextera ‘NPM PCR mastermix’ and amplified by PCR (first 72 °C 3 min, then 95 °C 30 s; then 22 cycles of: 95 °C 10 s, 55 °C 30 s, 72 °C 30 s, and finally 72 °C for 5 min). The five indexed libraries were then purified using AMPure XP magnetic beads (Beckman Coulter, #A63881) according to the manufacturer’s instructions. Finally, the library quality was assessed by evaluation using a high sensitivity bioanalyzer chip (Agilent, Santa Clara, CA, USA) and sequenced on a NextSeq 500 instrument (Illumina, San Diego, CA, USA). All primers are similar to those used in [12].

In total, 194 samples were sequenced in five BRB batches generating a total of 343 M reads. Batch effect was examined, and not observed. BRB-seq data was mapped by aligning reverse reads to the human reference genome (GRCh38.105) using STAR(2.7.9a) [29], then de-multiplexing by forward read barcoding using the BRB-seq tool 1.6.0, resulting in an average mapping rate of 70%.