Embryos electroporated for RNA sequencing analysis were dissected, dissociated, and EGFP-positive cells were isolated by fluorescence activated cell sorting at the Caltech Flow Cytometry Cell Sorting Facility as previously described (Hutchins et al., 2021 (link)). We prepared cDNA libraries from >1000 GFP-positive cells per replicate using SMART-Seq v4 Ultra Low Input cDNA Kit (Takara Bio) following manufacturer’s instructions. Libraries were then sequenced at the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory. 35 million 50bp single-end reads were collected on an Illumina HiSeq machine for each of two biological replicates of the dnBMPR1A-FLAG-expressing cranial neural crest cells. For differential analysis, RFP control (Hutchins et al., 2021 (link)) and BMP-inhibited reads were trimmed using Cutadapt (Martin, 2011 (link)), aligned to the GRCg6a chicken genome using BowTie2 (Langmead and Salzberg, 2012 (link)), transcripts were counted using FeatureCounts (Liao et al., 2014 (link)), and differential expression was determined using DESeq2 (Love et al., 2014 (link)). Resulting gene lists were analyzed using functional annotations in PANTHER (Mi et al., 2019 (link)).
Smart seq v4 ultra low input cdna kit
Manufactured by Takara Bio
The SMART-Seq v4 Ultra Low Input cDNA Kit is a laboratory tool designed for cDNA synthesis from low input RNA samples. It utilizes SMART (Switching Mechanism at 5' End of the RNA Transcript) technology to generate high-quality cDNA from as little as 10 pg of total RNA.
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Lab products found in correlation
2 protocols using smart seq v4 ultra low input cdna kit
1
Single-cell RNA-seq of BMP-inhibited neural crest
2
Transcriptome Analysis of Cranial Neural Crest Cells
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We used 1500 GFP+ cranial neural crest cells per replicate to prepare libraries. cDNA libraries were prepared using the Takara Bio SMART-Seq v4 Ultra Low Input cDNA kit, according to manufacturer instructions. RNA-Seq was performed at the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory at 35 million reads on two biological replicates for both the control cranial and Draxin overexpression cranial neural crest cells. Sequencing libraries were built according to Illumina Standard Protocols and SR50 sequencing was performed in a HiSeq Illumina machine by the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory. Sequence reads were aligned to the G. gallus genome (galgal6) with Bowtie2 (Langmead and Salzberg, 2012 (link)), transcript counts were calculated with HTSeq-Count (Anders et al., 2015 (link)), and differential expression analysis was performed with DESeq2 (Love et al., 2014 (link)). Gene lists were analyzed for functional annotation using PANTHER (Mi et al., 2019 (link)) and DAVID (Huang da et al., 2009a (link),b (link)).
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