GSM5068218: NTT V(D)J scRNA-seq; Mus musculus; RNA-Seq
mRNA was isolated using the Dynabeads mRNA DIRECT purification kit (Lifetechnologies) and was then amplified using the MessageAmp II aRNA Amplification Kit (Ambion) with the following modifications: in vitro transcription was performed at 37°C for 16h. First strand cDNA was synthesized using the Superscript III (Thermofisher) and a collection of TRAV/TRBV specific primers. TCRs were then amplified by PCR (20 cycles with the Phusion from NEB) with a single primer pair binding to the constant region and the adapter linked to the TRAV/TRBV primers added during the reverse transcription. A second round of PCR (25 cycles with the Phusion from NEB) was performed to add the Illumina adapters containing the different indexes. The TCR products were purified with AMPure XP beads (Beckman Coulter), quantified and loaded on the MiniSeq instrument (Illumina) for deep sequencing of the TCRa/TCRb chain. A Chromium Next GEM Chip G (10X genomics) was loaded with the appropriate number of cells, and the sequencing libraries prepared with the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 following the manufacturer's recommendations. Briefly, an emulsion encapsulating single cells, reverse transcription reagents and cell barcoding oligonucleotides was generated. After the actual reverse transcription step, the emulsion was broken, and double stranded cDNA generated and amplified in a bulk reaction. For 5' gene expression library this cDNA was fragmented, ligated to a sequencing adaptor, and PCR amplified. For V(D)J library preparation, a similar approach was followed except that 2 steps of PCR based V(D)J target enrichment were performed prior to fragmentation. Libraries were quantified by a fluorimetric method and their quality assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with 140 to 165 pM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 PE Cluster Kit reagents. Sequencing was performed on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents according to 10X Genomics recommendations (26 cycles read1, 8 cycles i7 index read, and 91 cycles read2).