title: Pooled CHO cells GEM well 1
design: Transfection of CHO cells with sample specific SBOs (42nM) with of 2.4 mg/ml PEI was performed 1 hour before to sample collection (i.e., the TM+ 8hr sample was transfected at 7hrs post TM treatment). Cells were counted and pooled following the cell sample preparation guide from 10X Genomics (CG00053  Rev C). Briefly, cells were centrifuged at 200 rcf for 3 minutes, the supernatant was removed and the cells were resuspended in PBS with 0.04% BSA with gentle mixing. Cells were passed through a 35 M strainer (Falcon cat. No. 352235) to remove cell debris and large clumps. Cell concentration was determined with the Luna TM automated cell counter and an equal number of cells from the different treatment conditions were pooled. Following sample pooling, the cell concentration was measured again (total conc. = 1180 cells/ul, viability = 94.3%) before to loading onto two GEM wells of a Chromium Chip. For the generation of cDNA and the matched SBO libraries (1 per GEM well), the 10X Genomics Single cell 3 v3.1 workflow (Chromium Next GEM Single Cell 3 Reagent Kits v3.1 CG000204  Rev D) was followed until the cDNA amplification step. At this stage the SBO additive primer (Table S2b) was to the cDNA amplification reaction at 0.015M final concentration. Following cDNA amplification, a clean-up step with 0.6X SPRI beads (Beckman Coulter, B23317) was performed according to the 10X Genomics protocol. The reverse transcribed SBOs are shorter than the cDNA libraries and were therefore present in the supernatant of the clean-up step, while the cDNA library derived from cellular RNA was retained on the beads. The cDNA libraries derived from cellular RNA were prepared following the 10X Genomics Single cell 3 v3.1 protocol (GC000204  Rev D). For the SBOs, the cDNA containing supernatant was transferred to a new tube and was further purified with two 2X SPRI purification steps. Briefly, 1.4X volume of SPRI beads was added to the SBO cDNA containing solution and following an 80% ethanol wash and elution step, an additional round of 2X SPRI beads purification was performed. The eluate was used for the SBO library preparation using a previously described protocol (Shin et al., 2019). The primers used for the 1st PCR for the amplification of the SBO libraries were: SBO-PCR-F and SBO-PCR1-R. For the second PCR the same forward primer (SBO-PCR-F) and two reverse primers SBO-PCR2-R1 or SBO-PCR2-R2 were used to differentiate between the samples loaded onto the two different Chromium chip lanes. The first PCR product was purified with a 1.8X SPRI bead purification step while the second PCR product was purified by size selection from an 8% acrylamide gel. Prior to sequencing concentration of all libraries was measured with the KAPA Library Quantification kit (KK4824) following manufacturers guidelines and library size was assessed with Tapestation traces (Agilent). The cellular RNA and SBO libraries were pooled and loaded onto two lanes of an Illumina NovaSeq configured to yield 28  91 bp reads. The mkfastq programme (10x Genomics Cell Ranger v6.0.2) was utilised to convert the resulting Illumina BCL files to FASTQ for further analysis.