title: Perturb_icCITEseq_hashtagoligo
library_construction_protocol: Approximately 1 million stained cells were resuspended with Cell Staining Buffer (BioLegend; #420201), transferred into a 15 mL Falcon tube and then pelleted by centrifugation for five minutes at 500 g. The supernatant was removed, leaving around 50 uL residual volume. Cell pellet was thoroughly resuspended in the residual liquid. Cells were fixed and permeabilized by dropwise addition of 1 mL pre-chilled True-Phos Perm Buffer (BioLegend, #425401) while vortexing and then incubated overnight at -20C. On the next day, fixed cells were equilibrated on ice and then centrifuged at 4C at 2000 g for five minutes. Pelleted cells were gently washed with ice-cold 2 mL of Intracellular Wash Buffer (1x) with 2 mM DTT and 0.2 U/uL Protector RNase Inhibitor (Roche, Cat#3335402001). The centrifugation step was repeated again to completely remove the supernatant. Intracellular staining was then performed using Intracellular Wash Buffer (1x) (BioLegend, custom part no. 900002577), with the addition of 2 mM DTT, TruStain FcX (BioLegend, #4223020), True Stain Monocyte Blocker (BioLegend, #426102) and 1 U/uL Protector RNase Inhibitor (Roche, #3335402001) in a 100 uL volume, as per manufacturer's recommendations. Stained cells were washed 3 times with 1 mL of Intracellular Wash Buffer (1x) and then resuspended and counted in Intracellular Wash Buffer (1x) with 2 mM DTT and 0.2 U/uL RNase inhibitor. A maximum of 4 uL of the cell suspension was used for processing with the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, Cat#1000268) and 3' Feature Barcode Kit (10X Genomics, Cat#1000262) according to the manufacturer's protocols. Use of over 4 uL will inhibit the reverse transcription reaction. TotalSeq-A hashtag and surface antibody libraries were prepared as per manufacturer's recommendations (BioLegend).