GSM7718434: 4T1, Day22, Fat, Pad, single cell 3; Mus musculus; RNA-Seq
Reverse transcription and cDNA amplification were carried out using a modified Smart-seq2 protocol (Picelli et al., 2014). Single cells were sorted by FACS as described above into 96-well plates containing 0.1 μl of RNase inhibitor (Clontech, Cat# 2313A), 1.9 μl of 0.2% Triton X-100 (Sigma, Cat# T9284), 1 μl of 10 µM oligo-dT primer (5′-AAGCAGTGGTATCA ACGCAGAGTAC T30VN-3′) and 1 μl of dNTP mix (10 mM each; Fermentas, Cat# R0192) in each well. Plates were either stored at -80 °C or processed immediately. Reverse transcription, PCR preamplification, PCR purification were performed following Picelli's protocol (Picelli et al., 2014). As mRNA quality control, 1 ul of each PCR product was used as template for qPCR detection of mCherry expression and samples with Ct value higher than 30 will be discard. PCR products were purified with 1 × Agencourt Ampure XP beads (Beckman Coulter, Cat# A63881) and the final product was reconstituted in 20 µl TE buffer. The concentration of cDNA was measured by Qubit™ 1× dsDNA High Sensitivity (HS) Assay Kit (Invitrogen, Cat# Q33230). Tagmentation and NGS library was carried out by using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, Cat# TD503) following the manufacturer's instructions. The purified library was qualify controlled by measuring the fragment size distribution with an Agilent HS DNA BioAnalyzer Chip and the concentration of library was measured with Qubit™ 1× dsDNA High Sensitivity (HS) Assay Kits following the manufacturer's instructions. Single cell cDNA libraries were sequenced on an Illumina HiSeq X Ten platform to obtain paired-end 150-bp reads. SMART-Seq2 (Picelli et al., 2014)