title: GSM6513570: batch_5, targeted mRNA; Homo sapiens; RNA-Seq
library_construction_protocol: Paired, fresh tumor and adjacent normal tissue samples were surgically removed from patients and immersed in MACS tissue storage solution (Miltenyi Biotec) immediately and kept ice-cold until the next step. Fresh samples were then washed with ice-cold phosphate-buffered saline (PBS) and then minced with the Iris scissor into small pieces, resuspended with digestion buffer consisting of RPMI-1640, DNase I (0.5 mg/ml, Sigma), collagenase II (1 mg/ml, Worthington) and collagenase IV (1 mg/ml, Diamond), incubated in 37 ℃ and gently piped until there were no obvious clumps. The cell suspension was further filtered through 70-µm nylon mesh to remove residues and cell aggregates, centrifuged at 300 rcf for 10 min, sediment was washed with wash buffer (RPMI-1640 with 10% FBS) and resuspended in 2 ml wash buffer. Density gradient centrifugation based on 20%-40%-70% Percoll gradient was performed for further purification of leukocytes. Purified cells were collected from the 40%-70% Percoll interface, and washed with ice-cold PBS. Red blood cells were excluded from the cell suspension using 1X Red Blood Cell Lysis Buffer (Invitrogen). Then the single-cell suspension was stained with anti-CD45 antibody (BD Biosciences) and Fixable Viability Dye eFluor (Biolegend) for the following cell sorting performed with SONY SH800 to obtain single alive immune cells. Single cells were carefully washed with BD Pharmingen Stain Buffer and labeled with distinctive sample tags following the manual of BDTM Human Single-Cell Multiplexing Kit. After incubation, labeled cells were then stained with viability markers (Calcein AM and Draq7), counted and pooled together at an equal ratio of all samples to obtain a total number of 40,000 live cells at most in 650 µl cold Sample Buffer. Mixed cells were loaded into primed and treated BD Rhapsody Cartridge fixed upon Express instrument, incubated at room temperature for 15 min to ensure the cells were randomly distributed into > 200,000 microwells of Rhapsody Cartridge. Cell Load Scan was performed with BD RhapsodyTM Scanner. Supersaturated Cell Capture Beads labeled with oligonucleotide barcodes were then loaded into the cartridge, incubated at room temperature for 3 min to capture the loaded cells. Cell-lysis was performed by incubation with freshly prepared Lysis Buffer, making for RNA fragments hybridized to the beads. Beads captured with mRNA were retrieved and washed with magnet and cold Bead Wash Buffer as soon as possible, following reverse transcription and then Exonuclease I incubation. To ensure that the intact V(D)J sequence can be read, optimal steps including template switching, denaturation, self-hybridization, and Klenow extension were needed before exonuclease treatment. Exonuclease I-treated beads were taken into libraries preparation following the BD Rhapsody system V(D)J CDR3 and Sample Tag library protocol. In brief, all beads were amplified in PCR1 with a targeted mRNA primer panel (including 399 immune-specific targets), sample tag primers, and TCR/BCR primers. The products were purified by AMPure beads for double-sided size selection and divided into shorter Sample Tag PCR1 products and longer targeted mRNA/TCR/BCR PCR1 products. Purified PCR1 products were then applied into separated PCR2 reactions with respective primers (targeted mRNA, sample tag, TCR, BCR). All the four PCR2 products were purified with AMPure beads for the following index PCR to label the sequences with distinct indexes. After another AMPure beads purification, the indexed products were the final sequencing libraries named targeted mRNA libraries, TCR libraries, BCR libraries, and Sample Tag libraries. Qubit Fluorometer was used to estimate the concentration of each library, and Agilent Bioanalyzer and qPCR were performed for quality control. Targeted mRNA libraries and Sample Tag libraries were sequenced by PE 150 (50,000 read pairs/cell, 600 read pairs/cell), while TCR libraries and BCR libraries were sequenced by PE 250 (1,000 read pairs/cell).