title: GSM8856300: Do-11-10_HybriSeq; Mus musculus; OTHER
library_construction_protocol: Round 3 PCR reactions were centrifuged at 500g for 1 minute to pellet cells. All round 3 PCR reaction solution was removed, pooled, and column purified with the Zymo DNA clean & concentrator kit (Zymo 11-305). Purified libraries were analyzed on an Agilent TapeStation Systems (D1000 kit) to check for the correct DNA size. If the predominate band was the correct size (252 ± 2 bp or 232 ± 2 bp depending if the left probe included a partial read 2 priming sequence) and was < 90% of the library the purified PCR product was run on a 3% agarose Tris-acetate-EDTA electrophoresis gel (200V 20 minutes) and the correct size band was cut out and extracted from the agarose with the Zymo Gel recovery kit (Zymo D4002). We observe that libraries that contained left probes containing the non-read 2 priming regions produced some nonspecific amplification requiring size selection purification. The purified pooled round 3 DNA product was placed into a final limited cycle PCR to add Illumina sequencing adaptors. The adapter addition PCR reaction was as follows: 0.5 ng DNA from pooled round 3 PCR product, 0.4 µM P7 forward primer, 0.4 µM P5 reverse primer and 1X KAPA HiFi HotStart ReadyMix. PCR thermocycling was performed as follows: 95 °C for 30 seconds, then 10 cycles at 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds, followed by a final extension at 72 ºC for 30 seconds. The PCR reaction was removed and purified with a 0.8X ratio of SPRI beads to generate an Illumina-compatible sequencing library. After probe ligation cells were counted and added to the ligase buffer (1X T4 DNA Ligase Reaction Buffer (NEB ref B0202S), 0.4 mM ATP, 40 U/mL RNasin, 0.5% Tween 20, 1% BSA, 200,000 U/mL T4 ligase) so that the final cell concentration was 22,000 cells/mL. Cells were passed through a 40 μm strainer. 22.69 µL of cells (500 cells) in ligase buffer were added to each well of a 96-well protein low bind plate, which had 2.31 µL of barcode 1 and linker 1 oligos already in each well. For the PBMC experiment, each stimulation condition was assigned to a specific set of wells, and this information was recorded. The first barcode was then used to identify the corresponding stimulation condition. Columns 1-4: Rest, columns 5-8: 6 hours, and columns 9-12: 24 hours. Cells were mixed by gently pipetting up and down. Plates were sealed and incubated at 25 ºC for 2 hours. 2 µL of 62.5 µM quenching oligo 1 (Supplementary table 6) were added to each well and mixed by pipetting. Plates were sealed and incubated at 25 ºC for 30 minutes. 25 µL of barcode wash buffer (50 mM EDTA, 0.5% Tween 20) was added to each well and incubated for 10 minutes. Cells from all 96 wells were pooled into a single 5ml low-bind Eppendorf tube. Cells were centrifuged for 3 minutes at 500 g at 4 ºC. Cells were washed two times in barcode wash buffer (+5 µM quenching oligo 1) and then washed in ligase buffer (+5 µM quenching oligo 1, -T4 ligase). Cells were resuspended in ligase buffer (+5 µM quenching oligo 1) so that the final cell concentration was 22,000 cells/mL and passed through a 40 μm strainer. 22.69 µL of cells in ligase buffer (+5 µM quenching oligo 1) were added to each well of a 96-well protein low bind plate, which had 2.31 µL of barcode 2 and linker 2 oligos already in each well. Cells were mixed by gently pipetting up and down. Plates were sealed and incubated at 25 ºC for 2 hours. 2 µL of 62.5 µM quenching oligo 2 were added to each well and mixed by pipetting. Plates were sealed and incubated at 25 ºC for 30 minutes. 25 µL of barcode wash buffer was added to each well and incubated for 10 minutes. Cells from all 96 wells were pooled into a single 5ml low-bind Eppendorf tube. Cells were centrifuged for 3 minutes at 500 g at 4 ºC. Cells were washed two times in barcode wash buffer (+5 µM each of quenching oligo 1 & 2) and then resuspended in ice-cold 1x ThermoPol reaction buffer (NEB ref B9004S) cells were passed through a 40μm strainer and counted. Cell concentration was normalized to 23,000 cells/mL in a cold ThermoPol reaction buffer. 460 cells were dispensed into 200µl PCR tubes or 96 well PCR plates. 20µL of PCR solution (1x KAPA HiFi HotStart ReadyMix (final concentration) and forward primer) with well-specific round 3 reverse primers (Supplementary Table 9) added to each well, so that the final concentration of each primer was 0.4 µM. PCR thermocycling was performed as follows: 95 °C for 30 seconds, then 20 cycles at 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds, followed by a final extension at 72 ºC for 30 seconds. For the probe tiling experiment 48 wells were used in each barcoding round with the final round 3 PCR barcoding reaction containing 115 cells.