GSM8164401: Erythroid_MULTIseq_cell_barcodes; Homo sapiens; RNA-Seq
Briefly, for each condition, 400,000 cells were rinsed twice in PBS and resuspended in 180 μl PBS. 20 μl of a 10X stock of anchor:barcode (per sample: 0.88 μl anchor, 0.44 μl 100 μM barcode, and 20.7 μl PBS) was added to each sample, pipetted gently to mix, and incubated on ice for 5 minutes. Next, 20 μl of a 10X stock of coanchor (per sample: 0.88 μl co-anchor and 21.2 μl PBS) was added to each sample, pipetted gently to mix, and incubated on ice for 5 minutes. 700 μl ice cold 1% BSA in PBS was then added to each sample, and cells were centrifuged at 4ºC for 5 minutes. The supernatant was aspirated and the wash was repeated 2X with ice cold BSA in PBS. After the last wash, cells were resuspended in 300 μl PBS/BSA, filtered through a cell strainer, and counted using a Nexcelom cell counter. Cells were adjusted to a concentration of 1 million/ml using ice cold BSA in PBS, and 50 μl of each sample was pooled into one tube and placed on ice. Cells were then loaded into the 10X Genomics microfluidics chip for processing and scRNAseq. For the ER-Hoxa9 experiment, 28,500 cells were loaded in order to obtain enough data for ~15,000 usable cells, and for the erythroid progenitor experiment, 22,000 cells were loaded in order to obtain enough data for ~12,000 usable cells (calculation performed at https://satijalab.org/costpercell/). Library construction proceeded as indicated in the 10X Genomics scRNAseq v3 protocol except as follows (adapted from McGinnis et al.). During cDNA amplification, 1 μl of a 2.5 μM MULTI-seq primer was added to the master mix. After the 0.6X SPRI clean-up, the supernatant was saved for barcode extraction rather than discarded. To the 60 μl supernatant, 260 μl SPRI beads and 180 μl 100% isopropanol were added for a final ratio of 1.8X beads, pipetted up and down 10x, and incubated at room temperature for 5 minutes. The tube was then placed on a magnetic rack for 5 minutes, supernatant discarded, beads washed 2x with 500 μl 80% ethanol, and eluted with 50 μl buffer EB. This SPRI purification step was repeated once more to further purify the barcode fraction. The barcode fraction was quantified using Qubit, and then library preparation PCR was performed using the Kapa HiFi HotStart ReadyMix with 3.5 ng barcode DNA, 2.5 μl 10 μM universal i5 primer, and 2.5 μl 10 μM TruSeq RPI1 primer in a 50 μl reaction for 8-12 cycles. The PCR product was purified using a final SPRI bead concentration of 1.6X, eluted, and quantified on a Bioanalyzer; a peak between 175 and 200bp was expected. The barcode library was mixed 1:10 with the transcript library and sequenced on an Illumina NextSeq 500.