GSM5946813: SPI2_mut_pool; synthetic construct; OTHER
RNA was extracted using RNeasy mini kit (Qiagen) with DNase-I treatment (45 min at 23°C). Resuspended RNA samples were treated with Turbo DNase (30 min at 37°C) for complete removal of bacterial DNA scPAIR-seq relies on the CEL-seq library preparation protocol (Hashimshony et al., 2016) up to in-vitro transcription (IVT) step. After IVT, amplified RNA (aRNA) was treated with EXO-SAP enzyme (15 min at 37°C) to remove unused primers leftovers. aRNA sample was split and half of the sample volume (11 µl) was used to perform nucleic acid purification with RNAClean XP beads. The purified aRNA was converted to cDNA using superscript Ⅲ with specific GFP primer and cDNA was purified with AMPure XP beads. MIB library was amplified by 25 PCR cycles using a nested GFP primer to improve specificity of MIB amplification. To ensure exclusive sequencing of MIB reads by a custom GFP primer in the downstream steps, the nested GFP-primer was attached with a 5′-tail containing Illumina 3′-adaptor that was modified in its binding site for the Illumina sequencing primer