title: GSM7623100: scSTAMP-seq_ErrorCorrection_Plate1_Lib3_Spatial; Homo sapiens; OTHER
library_construction_protocol: For FACS sorting, a single-cell suspension was made by dissociating cells using 0.25% trypsin-EDTA and inactivated using DMEM containing serum. Afterwards, cells were washed and resuspended in 1 mL of cold 1x DPBS-/- (without calcium or magnesium) (Gibco, 14040133) before being passed through a 35 µm nylon mesh cell strainer into a 5 mL round bottom polystyrene tube (Corning, 352235) that was then kept on ice. For plate-based single-cell capture, cell suspensions were FACS sorted for single cells into 384-well plates (Bio-Rad, HSP3801). For droplet-based single-cell capture, cell suspensions were processed for single cells by a 10X Genomics Chromium Controller following the workflow of the 10X Genomics Chromium Next GEM Single Cell 3' LT Kit v3.1 (10X Genomics, 1000325). scSTAMP-seq plate-based library preparation. 4 µL of Vapor-Lock (QIAGEN, 981611) was dispensed into each well of a 384-well plate using a 12-channel pipette. All downstream dispensing into 384-well plates was performed using a Nanodrop II liquid handling robot (BioNex Solutions). To each well, 100 nL of uniquely barcoded 7.5 ng/µL reverse transcription primers containing 6-nucleotide UMI was added. The reverse transcription primers used here have been described by Grün et al.33. Next, 100 nL of lysis buffer (0.175% IGEPAL CA-630, 1.75 mM dNTPs, 1:1,250,000 ERCC RNA spike-in mix (Ambion, 4456740), 0.19 U RNase inhibitor (Clontech, 2313A)) was added to each well. Single cells were sorted into individual wells of a 384-well plate using FACS. After sorting, plates were heated to 65°C for 3 minutes and returned to ice. Reverse transcription was conducted next by adding 150 nL of reverse transcription mix (0.7 U RNase OUT (Invitrogen, 10777-019), 2.34x first strand buffer (Invitrogen, 10777-019), 23.34 mM DTT (Invitrogen, 10777-019), 3.5 U Superscript II (Invitrogen, 18064-071)) to each well and the plates were incubated at 42°C for 1 hour and 15 minutes, 4°C for 5 minutes, 70°C for 10 minutes, and then held at 4°C. Next, second strand synthesis was carried out by adding 1.75 µL of second strand synthesis mix (1.74x second strand buffer (Invitrogen, 10812-014), 0.35 mM dNTP (Invitrogen, 10812-014), 0.14 U E. coli DNA Ligase (Invitrogen, 18052-019), 0.56 U E. coli DNA Polymerase I (Invitrogen, 18010-025), 0.03 U RNase H (Invitrogen 18021-071)) to each well and the plates were incubated at 16°C for 2 hours and then held at 4°C. 1 µL of nuclease-free water was added into each well to minimize losses during downstream pooling. The PHO, MPO, and endogenous mRNA molecules in a well now all contain the same RNA capture barcode, indicating a unique cell, which allows for pooling. Reaction wells receiving different barcodes were pooled using a multichannel pipette, and the oil phase was discarded. The small molecule spatial barcode portion was separated from endogenous transcript cDNA using a 3.2x SPRI bead cleanup with 1.8x 100% isopropanol in 2 steps. First, 0.7x AMPure XP beads (Beckman Coulter, A63881) were added and incubated at room temp for 15 minutes. At this step, it is critical to not dispose of the supernatant, as the spatial barcode library portion is found in the supernatant and the endogenous cDNA is bound to SPRI beads. The supernatant containing the spatial barcode fragments was then transferred to a fresh Eppendorf tube. The endogenous cDNA portion was prepared as described previously by Chialastri et al.31. Second, to the spatial barcode sample, an additional 2.5x beads and 1.8x 100% isopropanol (of the original pre-cleanup sample volume) were added and incubated at room temp for 15 minutes. The samples were placed on a magnetic stand for 5 minutes. The supernatant was removed and beads were washed twice with 80% ethanol. The sample was then eluted in 30 µL of nuclease-free water. The sample was then concentrated to 15 µL using an Eppendorf Vacufuge plus. Next, samples were prepared for PCR amplification by mixing 15 µL of the concentrated elute from the previous step, 25 µL of 2x PCR master mix (NEB, M0541S), 2 µL Illumina RPI primer (10 µM), 2 µL each of an 'inner' and 'outer' PCR primer (10 µM), and nuclease-free water up to 50 µL. This first PCR was performed at 94°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 10 cycles. A subsequent ExoSAP-IT treatment was then applied to cleanup the PCR product as follows. 2 µL of ExoSAP-IT (Applied Biosystems, 78200.200.UL) was added to 5 µL of sample from the previous PCR (for a total volume of 7 µL) and incubated at 37°C for 15 minutes. Another PCR was performed by mixing 7 µL of the cleaned-up PCR with 22.5 µL 2x PCR master mix, 2 µL Illumina RPI primer, 2 µL Illumina sample index primer, and nuclease-free water up to 45 µL. This second PCR was performed at 94°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 6-12 cycles.  Another ExoSAP-IT treatment was then applied to the 45 µL reaction by adding 5 µL of ExoSAP-IT to the previous PCR (for 50 µL total) and incubated at 37°C for 15 minutes. Finally, a 1.2x double-sided selection (0.90x left/0.30x right) was used to purify the spatial library, and a final ethanol wash was performed twice. The product was then eluted in 20 µL of nuclease-free water. Library fragment size distribution was determined using a 2100 Bioanalyzer. A schematic depicting each step of this process can be found in Supplementary Figure 1.  scSTAMP-seq droplet-based library preparation. Sequencing libraries were prepared using a custom protocol based on the 10X Genomics Chromium Next GEM Single Cell 3' LT Kit v3.1 (10X Genomics, 1000325) adapted from McGinnis et al.29. The 10X workflow was followed until cDNA amplification. We designed custom 'inner' and 'outer' PHO handle primers that allow scSTAMP-seq to be adapted for the 10X workflow (Supplementary Table 1). The scSTAMP-seq 10X 'inner' and 'outer' PHO primers were mixed to a working concentration of 2.5 µM. Next, the following cDNA amplification master-mix was made: 50 µL 10X amplification master mix, 15 µL 10X cDNA primer mix, 1 µL scSTAMP-seq 10X primers. Then, cDNA amplification was performed and an SPRI size selection was performed by incubating samples for 30 minutes with 0.6x AMPure XP beads to separate spatial and endogenous cDNA fractions. Next, the small molecule spatial barcode fraction was separated from endogenous transcript cDNA using the same strategy as described in the plate-based library preparation method above. The endogenous cDNA portion was then processed to generate Illumina libraries according to the 10X workflow and the spatial portion was processed to generate Illumina libraries as described in the plate-based library preparation workflow. A schematic depicting each step of this process can be found in Supplementary Figure 10.  scSTAMP-MAT-seq multiomic library preparation. The DNA accessibility and 5mC sequencing pipeline was performed as described previously in the scMAT-seq methods by Chialastri et al.31 up to the first SPRI bead clean up. At this step, the small molecule spatial barcode portion was separated from endogenous molecules using the same strategy as described in the plate-based library preparation workflow. The transcriptomic and epigenomic libraries were processed to generate Illumina libraries as described in scMAT-seq by Chialastri et al.31 and the spatial portion was processed to generate Illumina libraries as described in the plate-based library preparation workflow above. A schematic depicting each step of this process can be found in Supplementary Fig. 12.  Library sequencing was performed by Novogene on an Illumina HiSeq 4000 platform or at the Biological Nanostructures Laboratory (BNL) in the California NanoSystems Institute (CNSI) at the University of California Santa Barbara (UCSB) on an Illumina NextSeq 500 platform.