GSM5947040: inf_BMDMs 4 scPAIR-seq; synthetic construct; OTHER
Cells were harvested 20 hours post infection by a PBS wash followed by incubation of 10 min with ice-cold EDTA (5mM) and analyzed by flow cytometry (BD FACS Aria III) under continuous cooling at 4°C. Infected cells were sorted into 1.7ml low–bind tubes containing 25μl of lysis buffer (0.1% Triton in RNase-free water), placed immediately on dry ice and stored in minus 80. 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