GSM8198148: CROPseq_KO150_C2_ADT_HTO; Mus musculus; OTHER
Cells were washed with ice cold FACS buffer, scraped without trypsin, and centrifuged at 4 °C for 5 min at 500 rcf. The cells were resuspended in 100 µl FACS buffer with 1 µg TrueStain FcX PLUS (Biozym cat. No. 156604) per 1 million cells and incubated on ice for 10 minutes. Afterward, 100 µl FACS buffer containing CITE-seq antibodies (see Table S6; 1 µl per antibody per 10 million cells for KO15; 0.33 µl per 10 million cells for KO150) as well as TotalSeq-C hashing antibodies (BioLegend; 1 µl antibody per per 10 million cells) were added, and the incubation continued at 4 °C for 30 minutes. Cells were washed twice with FACS buffer and resuspended in fresh FACS buffer with Zombie NIR viability staining (BioLegend cat. No. 423105) at a 1:1000 dilution. Cells were sorted on a FACS Aria by selecting NIR negative (live) cells that were mCherry positive. The replicates of the same condition for the KO150 library were sorted, with 50,000 cells each going into three tubes. Superfluous supernatant was reduced using the VRNxT machine and processed according to the Chromium Next GEM Single Cell 5' v2 Dual Indexing kit with Feature Barcoding (10x Genomics cat. No. 1000263), with the following modifications. At the GEM generation, step 1.1, an additional 0.5 µl of a 10 µM stock CROPseq-3P5P_gRNA_RT primer (5'-AAGCAGTGGTATCAACGCAGAGTACCAAGTTGTTAACGGTCTAGCC-3') were added to the master mix.  To limit technical bias/batch effects we sorted each of the three RAW264.7 thaws (individually hashtagged) together (1:1:1) for a total of three tubes per treatment (3 tubes x 3 treatments = 9 samples). One tube per treatment was processed on a 10x chip resulting in a total of 3 chips being used. The treatment is indicated by the letter in the library name, the chip/tube number is indicated by the number (e.g. "_A1_"). The cDNA amplification was performed for 11 cycles, and during the SPRI select clean-up, the remaining 80 µl supernatant were mixed with 30 µl beads to perform a 1.2x clean-up, otherwise following the 2.3B Cell Surface Protein protocol. 5 µl of the guide RNA fraction were amplified with the following PCR mix: 3 µl of a 10 µM fwd/rev CROPseq-3P5P i7/i5 primer mix (see Table S6), 2x Kapa HIFI MM (Roche cat. No. 7958935001), 0.5 µl SYBR green (Life Technologies cat. No. S7563) and 16.5 µl water. The PCR was run for 30 sec at 98 °C, 7 to 8 cycles of (10 sec at 98 °C, 30 sec at 70 °C and 20 sec at 72 °C), followed by 1 min at 72 °C. The amount of amplification cycles was determined by inspecting the fluorescence signal. The reaction was stopped once the amplification has reached the early exponential phase. A left-sided 0.9x SPRI bead clean-up was performed, and the DNA was eluted in 15 µl EB buffer. Correct amplification was determined using a Bioanalyzer HS chip, confirming a peak around 272 bp for the guide RNA library. For the KO15 and KO150 experiments, cell surface and guide RNA libraries were added to the gene expression library at 10% and 15% of the final concentration, respectively.