RNAprotect Cell Reagent (250 ml)

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Publication protocol

RNA was stabilized by resuspending cells in RNAprotect Cell Reagent (QIAGEN) and RNase inhibitors (Promega). Prior to FACS sorting, cells were diluted in PBS (Invitrogen). Single cells were sorted into 5 μl lysis buffer consisting of a 1/500 dilution of Phusion HF buffer (New England Biolabs) and ERCC spike-ins (Ambion), spun down and frozen at −80°C. Plates were thawed and libraries prepared as described previously (Soumillon et al., 2014). Briefly, RNA was desiccated after protein digestion by Proteinase K (Ambion). RNA was reverse transcribed using barcoded oligo-dT primers (IDT) and products pooled and concentrated. Unincorporated barcode primers were digested using Exonuclease I (New England Biolabs). Pre-amplification of cDNA pools were done with the KAPA HiFi HotStart polymerase (KAPA Biosystems). Nextera XT libraries were constructed from 1 ng of pre-amplified cDNA with a custom P5 primer (IDT).

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