GSM2579604: Fcx1_scTHS-Seq; Homo sapiens; OTHER
Cryofrozen post-mortem brain tissue was treated with nuclear extraction buffer, isolated nuclei was stained with DAPI and FACS purified. To the 384 well plate of freshly generated uniquely barcoded Tn5059 r5 transposome complexes, 4 µL of human/mouse mixed cell sample was added for a total of ~960 nuclei/well and final concentration of 0.7 µM Tn5059 r5 transposome complex. Each sample was mixed gently 5X by the electronic pipettor. Optimally ~2000 nuclei/well are wanted, however nuclei yields were low. The 384 well plate was then incubated at 37ºC for 30 minutes. To stop the reaction, 4.0 µL of 50 mM EDTA was added to each well and mixed gently 5X by the electronic pipettor for a final concentration of 20 mM EDTA in 10 uL, followed by incubating the 384 well plate at 37ºC for 15 minutes. The 384 well plate of samples was then frozen at -20ºC overnight. The next day, the 384 well plate of samples was thawed, and one volume of cold 2X FACS buffer (1X FACS buffer: 2 mM EDTA, 1% BSA in PBS) was added to each well. Samples are then mixed gently 3X by the electronic pipettor and pooled into one tube on ice, which was spun down at 500xg for 5 minutes at 4ºC. Supernatant was removed, and tagmented nuclei resuspended in 1.5 mL cold 1X FACS buffer. Next, 75 µL eBioscience Propidium Iodide (PI) was added to the tube, and gently mixed. Nuclei were then sorted by flow cytometry into 96 well plates containing 10 µL PBS/well at 100 nuclei/well, and put on ice. Doublets were removed based on forward and side scatter plots, and PI stained events selected. Nuclei were now ready for RNA amplification and processing. Each 96 well plate of nuclei was processed individually. First, 11 µL guanidine hydrochloride was added to each well and mixed by lightly vortexing. Reactions were purified by addition of 40 µL (1.8X) AMPure SPRI beads and lightly vortexed, then bead pelleting and 80% ethanol washes were performed with the “flick and blot” method and magnetic plate from V&P Scientific. After 80% ethanol washes were complete the plate was quickly spun down at 500xg and leftover 80% ethanol removed by pipetting. 10 µL 1X NEB Taq polymerase was added to each reaction and the plate was lightly vortexed to resuspend the beads (SPRI beads left in reaction), followed by running the reactions at 72ºC for 3 minutes for end fill in and put on ice. For in vitro transcription (IVT) amplification the NEB HiScribe T7 high yield synthesis kit was used. To each reaction a mastermix of 2 µL of 10X transcription buffer, 2 µL ATP, 2 µL CTP, 2 µL GTP, 2 µL UTP was directly added to the end fill in reactions, lightly vortexed, and incubated at 37ºC for 19 hours. To check IVT amplification, two full reactions were purified with SPRI binding buffer, run on a 6% Tris-Borate-Urea (TBU) gel for 20 minutes at 250 volts followed by SYBR gold staining and visualization on a UV-transilluminator. Next, reactions were purified by addition of 44 µL (2.0X) SPRI binding buffer (20% PEG 8000, 2.5 M NaCl, 10 mM Tris–HCl, 1 mM EDTA) to each reaction and the plate vortexed thoroughly. 80% ethanol washes and leftover 80% ethanol removal were performed as describe above, and SPRI beads were resuspended in 9 µL nuclease free water. For reverse transcription, first 2.5 µL of 20 µM random hexamers was added to each reaction, the plate vortexed lightly and then heated to 70ºC for 3 minutes, and immediately cooled on ice. Then Clontech SMART® MMLV Reverse Transcriptase kit was used with addition of 4 µL 5X first strand synthesis, 2 µL dNTP mix, 2 µL 100 mM DTT, 0.5 µL SMART MMLV RT in a mastermix to each reaction and the plate vortexed lightly. Reactions were incubated at 22ºC for 10 minutes, then 42ºC for 60 minutes, and terminated at 70ºC for 10 minutes. To degrade RNA in cDNA-RNA hybrids 1 µL of 0.5 units Enzymatics RNase H was added to each reaction, the plate was vortexed lightly and incubated at 37ºC for 20 minutes. For second strand synthesis first 2.5 µL of 20 µM sss_scnXTv2 (Table S12) was added to each reaction and lightly vortexed, then incubated for 2 minutes at 65ºC and immediately cooled on ice. Then 5.9 µL of NEB taq5X was added to each reaction and incubated at 72ºC for 8 minutes. After cooling on ice, 60 µL (2.0X) SPRI binding buffer was added to each sample. The plate was vortexed thoroughly and 80% ethanol washes and leftover 80% ethanol removal performed as described previously, and SPRI beads resuspended by light vortexing in 7 µL of nuclease free water. Double stranded cDNA fragments then undergo simultaneous fragmentation and 3' adaptor addition with a custom nXTv2_i7 Tn5059 transposome (Table S12). To 7 µL of sample, 2 µL of 5X tagmentation buffer was added to each sample, followed by addition of 2 µL of prepared 0.7 µM custom nXTv2_i7 Tn5059 transposomes (final transposome concentration of 0.14 µM), vortexed lightly, then incubated at 55ºC for 6 minutes, and cooled briefly on ice. Immediately after cooling, 19 µL of 6.32M guanidine hydrochloride, for a final guanidine hydrochloride concentration of 4M was added to each reaction and briefly vortexed. Then 60 µL (2.0X) SPRI binding buffer was added to each sample. The plate was vortexed thoroughly and 80% ethanol washes and leftover 80% ethanol removal was performed as described previously. However, for this purification SPRI beads were resuspended in 16 µL nuclease free water and the plate was placed back onto the magnetic plate. Sample was eluted off SPRI beads held by the magnetic plate, and transferred to a qPCR plate. Standard Illumina Nextera XT v2 barcoding in an 8x12 (i5xi7) format was performed with qPCR, using custom scTHS-seq i5 indexes and standard Illumina i7 indexes (Table S12). In total, 20 µL of KAPA SYBR Fast, 2 µL of 10 µM scT7_S5XX index primer and 2 µL of 10 µM nXTv2_i7XX index primer were added to each reaction for a total volume of 40 µL, and mixed well. qPCR was run at 72ºC for 3 minutes, 95ºC for 30 seconds, followed by cycling for (95ºC for 10 seconds, 63ºC for 30 seconds, 72ºC for 1 minute) until curves reach saturation, typically 9-12 cycles. Plates were stored at -20ºC. scTHS-seq and Nextera
GEO Accession
GSM2579604