title: GSM7257027: H9FF polCA7_distal (batch with mutant samples of GSE175592); Homo sapiens; RNA-Seq
library_construction_protocol: All the polCAs and control (conCAs) have been manually dissected at a stereomicroscope and processed for RNA extraction. For bulk RNAseq, samples were lysed in 350 µl of RLT buffer supplemented with βmercaptoehtanol and RNA was extracted using the RNeasy Micro Kit (Qiagen 74004) following the manufacturer's protocol. For scRNAseq, manually dissected at a stereomicroscope and processed for dissociation with gentleMACSTM Dissociator in Trypsin/Accutase solution with TURBO™ DNase (Thermo AM2238, 2 U/µL). After dissociation, DPBS-/- + 10% FBS has been gradually added to stop reaction. Samples have been then centrifuged at 400G for 5min at 4ºC and supernatant was aspirated without touching the pellet. The pellet has been resuspended in additional  500µl of DPBS-/- + 10% FBS, filtered through FACS tubes and stained for viability dye DRAQ7 (Biostatus, DR70250, 0.3mM). Live cells have been sorted with a BD FACS Aria™ III on Alexa 700 filter and barcoded with unique CMOs for MULTI-seq analysis. For bulk RNAseq, we then assessed RNA concentration and quality with an RNA 6000 Nano Chip (Agilent Technologies) and enriched for mRNA with NEBNext Poly(A) mRNA Magnetic Isolation Module; barcoded samples were then multiplexed and sequenced 50 bp single-end on a HiSeq 2500 (Illumina). mRNA sample enrichment, library preparation, and sequencing were performed by the VBCF NGS Unit (https://www.vbcf.ac.at).