Single Cell scale RNA-seq Analysis Protocol to analyze Smart-3SEQ data from RAGP neurons of pig heart

Abstract The present protocol describes transcriptome mapping, data normalization and analysis pipeline with detailed steps for each of these aspects for single cell/ low input RNASeq data from Right Atrial Ganglionated Plexus (RAGP) of pig heart. The protocol with minor modifications can be adapted for low input samples with short reads or samples with low quality input RNA. Single cell samples acquired using Laser Capture Microdissection (LCM) were processed for RNA-Seq library preparation using Smart-3SEQ technique (Foley et al 2019). The data analysis workflow consists of (a) pre-processing- data trimming, read alignment and feature count and (b) downstream analysis- annotation, batch correction, filtering and normalization. The entire protocol is performed using freely available packages. Most of them are available within the R framework.