GSM5819679: RNAseq_CD4_tNGFR_stim_3; Homo sapiens; RNA-Seq
For the screen, gDNA was extracted using GeneJET DNA Purification Kit (Thermo Fisher Scientific). For the single cell experiment, isolations were performed according to manufacturer's protocol. For bulk RNAseq, total RNA was extracted using the Direct-zol RNA purification kit (Zymo). For ATAC-seq, cell membranes were lysed in the RSB buffer (10mM Tris-HCL pH 7.4, 3mM MgCl2, 10mM NaCl) with 0.1% IGEPAL freshly added. After pipetting up and down, nuclei were isolated by centrifugation at 500g for 5 minutes at 4°C. After discarding the supernatant, the nuclei were resuspended in the Tagmentation DNA (TD) Buffer with homemade transposase TnY protein and incubated at 37°C for 30 minutes, and purified on a MinElute column (Qiagen). For the screen, two rounds of PCR to amplify ORF barcodes and add Illumina adaptors were performed on gDNA.   For the single cell experiment: cDNA amplification, additive primers for amplification of sample hashing and surface antigen barcodes were included, as well as a nested reverse primer binding to the puromycin resistance cassette downstream of the ORF. Following cDNA amplification, SPRI beads were used for size selection of resulting PCR products – small size (<300 bp) sample hashing and surface antigen barcodes were physically separated from larger cDNA and ORF amplicons for downstream processing. Sample hashing and surface antigen barcodes were also processed. Amplified cDNA was then separated into three conditions, for construction of the gene expression library, alpha beta TCR library and ORF library. The ORF library was processed similarly to alpha beta TCR library, using nested reverse primers binding downstream of the ORF. The quality of produced libraries was verified on BioAnalyzer using High Sensitivity DNA kit (Agilent). The libraries were sequenced on NextSeq 500. For the gene expression library, >25,000 reads per cell were generated. For other libraries, >5,000 reads per cell were generated.  For bulk RNAseq, RNA was reverse transcribed using SMARTScribe Reverse Transcriptase (Takara Bio) and a poly(dT) oligo containing a partial Nextera handle. Resulting cDNA was then PCR amplified for 3 cycles using OneTaq polymerase (NEB) and tagmented for 5 minutes at 55C using homemade transposase TnY. Immediately afterwards the tagmented DNA was purified on a MinElute column (Qiagen) and PCR amplified using OneTaq polymerase and barcoded primers for 12 cycles. The PCR product was purified using a dual (0.5X-0.8X) SPRI cleanup (Agencourt) and the size distribution was determined using Tapestation (Agilent). Samples were sequenced on a NextSeq 500 (Illumina) using a v2.5 75-cycle kit (paired end).  For ATAC-seq, the tagmented DNA was PCR amplified using a homemade Pfu X7 DNA polymerase and barcoded primers for 12 cycles. The PCR product was purified via a 1.5X SPRI cleanup (Agencourt) and checked for a characteristic nucleosome banding pattern using TapeStation (Agilent). Samples were sequenced on a NextSeq 500 (Illumina) using the v2.5 75-cycle kit (single end).