title: GSM8420771: XX_D4_MULTIseq_rep1_dFtxXert; Mus musculus; RNA-Seq
library_construction_protocol: For sample multiplexing, MULTI-seq barcode-lipid complexes were used according to the published protocol with minor modifications(McGinnis et al. 2019). Sample barcodes were chosen from a list of compatible barcodes(Rosen et al. 2022). Lipid-modified anchor and co-anchor oligos were purchased from Sigma-Aldrich. Barcode- and library preparation oligos were purchased from Eurofins with NGSgrade quality. Per sample, 1*105 cells were transferred to a 96-well ultra-low attachment plate (Costar) and kept on ice during the procedure. After two washes with PBS, the cells were incubated 5-10 min with an Anchor:Barcode solution (45 µl PBS, 5 µl Anchor:Barcode). The mixture was incubated for another 5-10 min after adding 5 µl of Co-Anchor solution. Labeling was quenched by adding 1% BSA in PBS, samples washed once in the same solution and resuspended in 0.4% BSA in PBS. Afterwards, cells were counted, pooled equally, and filtered using a Flowmi cell strainer 40 µm (Sigma-Aldrich). 2.5*104 cells were used for scRNA-seq gene expression library preparation . The 10x Genomics library preparation protocol was performed according to manufacturer's instructions (CG000315 Rev E, 10X Genomics) with the additional steps required to generate the separate MULTI-seq libraries for the sample-to-cell barcode assignment(McGinnis et al. 2019). During cDNA amplification, 0.5 µL of 1.25 μM “MULTI-seq additive primer” was added. 50% of the purified gene expression cDNA was carried forward, and the cycle number in the index PCR was reduced by one accordingly, to increase library complexity(Bolondi et al. 2024). During the first step of cDNA cleanup the supernatant was kept, which contained the short MULTI-seq library. This MULTI-seq library was then cleaned up two times using SPRI beads (KAPA HyperPure Beads, Roche). Sequencing adapters containing indexes (“TruSeq_RPIX_”, and “Universal_I5_with_index”) were added by PCR, using 2.5 µL primer at 10 µM each, 10 ng of cDNA, 26.25 µL Kapa HiFi HotStart ReadyMix 2x, and water to 50 µL final volume, with a program of 95 °C 5 min, 11 cycles of 98 °C 15 sec, 60 °C 30 sec, 72 °C 30 sec; followed by 72 °C 1 min and 4 °C hold. The resulting sequencing library was then cleaned up another time with SPRI beads. 10X gene expression libraries and MULTI-seq libraries were sequenced asynchronous 90/28 bp paired-end on the NovaSeq 6000 platform.