GSM7688460
RNA was extracted using TRI reagent (Sigma GmBH, Mannheim, Germany) in combination with PureLink column-based kit (Thermo Fisher, Waltham USA). RNA was quantified using Nanodrop 2000 (Thermo Fisher, Waltham, USA) and RNA integrity was confermed using Tapestation. 10ng was used for library creation for MarsSeq sequening. A bulk adaptation of the MARS-Seq protocol (Jaitin et al., Science, 2014; Keren-Shaul et al., Nature Protocols, 2019) Briefly, 30 ng of input RNA from each sample was barcoded during reverse transcription and pooled. Following Agencourct Ampure XP beads cleanup (Beckman Coulter, Indianapolis, IN), the pooled samples underwent second strand synthesis and were linearly amplified by T7 in vitro transcription. The resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with Illumina sequences during ligation, RT, and PCR. Libraries were quantified by Qubit and TapeStation.