Instrument: HiSeq X Ten
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA of each tissue was purified using standard TRIzol based protocols for bulk RNA-seq; For single-nucleus lysate of single cell ATAC-seq, tissues were simply rinsed with 0.8X PBS, and dried tissues were snap-frozen in a clean mortar with liquid-nitrogen, fragile tissues were then grounded into powder and transferred into 1ml of ice-cold lysis buffer (0.1% IGEPAL CA-630 (Sigma-ALDRICH), 0.01% digitonin (Stemcell), 0.1% Tween-20 (Diamond), 1%BSA,10mM Tris-HCL pH7.5 (Thermo Fisher Scientific), 10mM NaCl (Sangon Biotech), 3mM MgCl2 (Thermo Fisher Scientific) in DEPC Treated Water (Sangon Biotech)). The lysis was performed on ice for 3min, and 5ml of RSBT (0.1% Tween-20, 1%BSA, 10mM Tris-HCL pH7.5, 10mM NaCl, 3mM MgCl2 in DEPC Treated Water) was added, the medium was then filtered through a 40μm strainer to remove the debris and clumps for each sample. Nuclei were washed with RSBT for 1 time and pellet was resuspended with 5ml 1X PBS, after which 140ul of 37% formaldehyde solution (HUSHI) was added to a final concentration at 1%. Nuclei were incubated at room temperature for 10min, then 250ul 2.5M Glycine (Diamond) was added to quench the reaction at the same condition for 5min, and finally the mixture was placed on ice for 15min to stop cross-linking completely. After washing with 1ml RSBT for 1 time, nucleus pellet could be resuspended with freezing buffer (50mM Tris-HCL pH8.0 (Thermo Fisher Scientific), 25% Glycerol (Sangon Biotech), 5mM Mg (OAc)2, (Sangon Biotech), 0.1mM EDTA (Thermo Fisher Scientific) in DEPC Treated Water). For bulk RNA-seq, RNA extraction followed reverse transcription, second strand DNA synthesis, transposase tagmentation and standard Illumina Nextera library construction. For single-cell ATAC-seq, library preparations were performed with the combinatorial hybridization seq protocls, 96-384 uniquely indexed Tn5 transposase were assembled prior to tagmentation. For each well, the Tn5 primers (25pM Tn5 primer A oligo, 50pM Tn5 ME oligo, as well as 25pM the barcoded Tn5 primer B oligo) were mixed, then the plate was incubated at 95℃ for 2min and slowly cooled to 10℃ with a temperature ramp of −0.1℃/s. Tn5 transposase (Vazyme Biotech) was diluted and mixed with annealed Tn5 primers, and the mixture was then incubated at 30℃ for 60min,which leads to a final indexed Tn5concentration of 40ng/ul. Frozen nuclei in freezing buffer were thawed at 37℃ and centrifuged at 500g for 5min. Thawed frozen nuclei or fresh fixed nuclei were resuspended with 1ml RSBT and filtered with a 40um cell strainer to remove any clumps during cryopreserving. Nuclei was centrifuged at 500g for 5min and resuspended in tagmentation buffer (10mM Tris-HCl pH7.5, 5mM MgCl2, 10% DMF (Sigma-ALDRICH), 1%BSA, 0.1% Tween-20, 0.01% digitonin and 0.4X PBS). For each tissue, nuclei density was determined using a blood cell counting chamber. Nuclei were then split into 96-well plates. For each well, 23.5ul nuclei (10000-15000 nuclei) was mixed with 1.5ul indexed Tn5. Then the tagmentation was carried out at 55℃ for 30min, and 25ul 2X stop buffer (40mM EDTA,1mM Spermidine) was separately added into 96 wells. The plate was incubated at 37℃ for 15min to completely stop tagmentation. All the reagent was pooled together into a 15ml centrifuge tube and then centrifuged at 500g for 5min. The pellet was washed twice with RSBT before split into new 96-well plates. Hybridization buffer within cells were split into eight 96-well plates (3ul for each well) and 2ul 25μM pre-annealing hybridization primers (50μM HY head oligo,50μM barcoded HY primer oligo, mixed equally and incubated at 95℃ for 2min, then slowly cooled to 25℃ with a temperature ramp of −0.1℃ /s) were added to each well. Plates were incubated at 37℃ for 90 min and 0.5ul 100μM block tail primer oligo was added to block any redundant hybridization primers. After blocking for 30min at 37℃, all reagents were pooled into a 15ml centrifuge tube and then centrifuged at 500g for 5min. Pellet was washed with inPBS twice and resuspended with 40ul inPBS. Then 60ul PNK mix (10ul 10X PNK buffer (NEB), 20ul T4 Polynucleotide Kinase (NEB), 10ul 10mM ATP (NEB), 20ul DEPC treated water) was added. The PNK reaction was carried out at 37℃ for 30min. After PNK reaction, 1ml ice-cold RSBT was added in pooled reagent were then filtered using a 40μm cell strainer. The medium was centrifuged at 500g for 5min. After discarding the supernatants, the pellet was again resuspended with RSBT. The density of nuclei was estimated and nuclei were split into new 96-well plates (5000-8000 nuclei per well). The volume of each well was adjusted to 8ul. Then 2ul gap-fill mix (1ul 10X Ampligase DNA ligase buffer, 0.3ul Ampligase DNA ligase (Lucigen), 0.5ul 10mM dNTP, 0.2ul T4 DNA polymerse (NEB)) was then added into each well. The plates were incubated at 37℃ for 60 min. Then 2ul nuclei lysis buffer (1ul 10mM Tris-HCl pH8.0,0.5ul proteinase K,0.5ul 1%SDS) was added to release the fragments in nuclei by incubating at 55℃ for 60 min. For each well, the mixture was then purified using 1.5X VAHTS DNA Clean Beads. Then 10ul product in DEPC treated water was then transferred into a new 96-well plate. For each well, 14ul PCR mix (12ul 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 1ul 10mM P5 primer,1ul 10mM indexed P7 primer) were added to the plates. The PCR program was as follows: 72°C for 5 min; 98°C for 3 min; 12 cycles of 98°C 10 s, 65°C 15 s, and 72°C 1 min; 72°C 5 min and 4°C hold. After pooling PCR products, 1.0X VAHTS DNA Clean Beads were used to purify the DNA library, the concentration of dsDNA was quantified. The purified linear DNA library was circularized into single strand DNA (ssDNA) library using VAHTS® Circularization Kit for MGI (Vazyme). Then ssDNA library was amplified using DNBSEQ DNB preparation kit (MGI). Amplified DNA nanoball (DNB) was sequenced with custom primers on MGI DNBSQ-T7 with dark reaction model ( CH-ATAC-seq: 100 cycles of read1 with dark reaction from 11-33,44-62, 100 cycles of read2).