Drop-seq was performed as described previously (Macosko et al., 2015 (link); Xie et al., 2017 (link)) with small modifications. Briefly, droplets were generated using microfluidic devices, which encapsulated single cells and barcoded beads (ChemGenes Corporation, Wilmington, MA, catalog number Macosko201110). Beads were collected after the breakage of individual droplets using perfluorooctanol (Sigma-Aldrich, St. Louis, MO, catalog number 370533). RNA hybridized on the barcoded beads was reverse transcribed using Maxima H minus reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, catalog number EP0751) and cDNA was amplified with 13 cycles (KAPA HotStart ReadyMix, Kapa Biosystems, Wilmington, MA, catalog number KK2602). In-house Tn5 transposase was used to insert sequencing adapters and fragmentate cDNA. Illumina Nextera adapters i5 and i7 were used to amplify the fragmented cDNA with 12 cycles (KAPA HiFi PCR Kits, catalog number KK2102). Agarose gel size selection was performed to recover fragments with length of 400 to 600 bp.
Maxima h minus reverse
Manufactured by Thermo Fisher Scientific
Maxima H minus reverse is a high-performance reverse transcriptase enzyme designed for cDNA synthesis. It catalyzes the conversion of RNA to cDNA, which is a crucial step in various molecular biology applications such as gene expression analysis, qRT-PCR, and RNA sequencing.
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2 protocols using maxima h minus reverse
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Single-Cell RNA Sequencing Using Drop-seq
2
Reverse Transcription and qPCR Analysis
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Reverse transcription (RT) was carried out using 5 μg of total RNA extracted from the respective cells as described above, 100 pmol of random hexamer primer and 100 U of Maxima H Minus reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s protocol. PCR with the obtained cDNA was performed on LightCycler 96 (Roche) using SYTO9 fluorescent dye (Thermo Fisher Scientific), HS-Taq polymerase (Biolabmix) and gene-specific primers (Supplementary Table S3 ). The experiments were carried out in three biological replicates. Relative quantification of expression levels of genes of interest was determined by normalizing them to that of the GAPDH gene using the LightCycler 96 integrated software. The data obtained were analyzed by GraphPad Prism version 8.3.0 using the Mann–Whiney test. Pearson’s correlation between the Log2FoldChage (LFC) values of the tested gene set calculated in Excel from the RT-qPCR data and the LFC values determined for the same genes from the RNA-seq data analysis (see below) was estimated using the cor.test function from the R Stats package.
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