title: GSM8842411: sgAcat1_EV_rep2_R1; Mus musculus; RNA-Seq
library_construction_protocol: For Smart-seq2 (switching mechanism at 5' end of the RNA transcript), single NK cells were sorted and processed as described in previous study. Briefly, total RNA was extracted and mixed with anchored oligo-dT primer (10 µM, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, where “N” is any base and “V” is either “A”, “C” or “G”) and dNTP mix (10 mM), denatured at 72 °C for 3 min and placed on ice immediately. For RNA-seq, total RNA was extracted with Trizol reagent (Invitrogen) and mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. For Smart-seq2，7 µL of the first-strand reaction mix, containing 0.50 µL SuperScript II reverse transcriptase (200 U/µL), 2 µL Superscript II First-Strand Buffer (5×), 0.25 µl RNAse inhibitor (40 U/µL), 0.9 µL MgCl2 (100 mM), 0.25 µL DTT (100 mM), 2 µl betaine (5 M), 1 µL TSO (10 µM) and 0.1 µL nuclease-free water were added for reverse transcription. After this, samples were followed by PCR preamplification with the mixture containing: 25 µL KAPA HiFi HotStart ReadyMix, 1 µL ISPCR primers (10 µM, 5′-AAGCAGTGGTATCAACGCAGAGT-3′) and 14 µL nuclease-free water (Gibco). Finally, after tagmentation reaction and PCR amplification, samples were sequenced on Illumina HiSeq 2000. For RNA-seq, Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. High-throughput sequencing was performed on an Illumina Novaseq platform and 150 bp paired-end reads were generated.