GSM5068564: TIL_sc-seq - ADT/HTO; Mus musculus; RNA-Seq
Tumors were harvested, processed and FAC sorted for TCRB+CD3+CD90.2+CD11b- cells followed by ADT and HTO tagging and droplet encapsulation for single cell sequencing using 10xGenomics platform All samples were prepared using the 10x Genomics Single Cell 5′  protocol according to the manufacturer's instructions up until the cDNA amplification step (10x Genomics, USA). Two picomoles of HTO and ADT additive oligonucleotides were spiked into the cDNA amplification PCR, and cDNA was amplified according to the 10x Single Cell 3′ v3 protocol (10x Genomics, USA). Following cDNA amplification, 0.6X SPRI was used to separate the large cDNA fraction derived from cellular mRNAs (retained on beads) from the ADT- and Cell Hashtag (HTO)-containing fraction (in supernatant). The cDNA fraction was processed according to the 10x Genomics Single Cell 3′ v3 protocol to generate the transcriptome library; indexing was done using Chromium i7 Multiplex Kit. An additional 1.4X reaction volume of SPRI beads was added to the ADT/HTO fraction to bring the ratio up to 2.0X. The beads were washed with 80% ethanol, eluted in water, and an additional round of 2.0X SPRI performed to remove excess single-stranded oligonucleotides from cDNA amplification. After final elution, separate PCRs were set up to generate the CITE-seq ADT library (SI-PCR and RPI-x primers) and the HTO library (SI-PCR and D7xx_s). Standard 10X genomics library prep