GSM5862108: Pdzk1ip1-in vivo-wk3 tp wk5 RNA; Mus musculus; RNA-Seq
Bulk RNA-Seq: RNA from sorted cells was extracted using TRIzol Plus purification kit (ThermoFisher) and cDNA libraries were prepared with the Low input Clontech SMART-seq HT with Nxt HT.  Flip-Jump-Seq (MiSeq): Genomic DNA was extracted by NucleoSpin Tissue XS (Takara) (< 15,000 cells) or QIAamp Mini  kit (> 15,000 cells). Extracted DNA was further amplified with REPLI-g Mini Kit (Qiagen) for whole genome amplification (WGA).Three-arm LM-PCR-based method was optimized and adapted to clone FlipJump transposon integration sites from both 3TR and 5TR ends. Amplified DNA was quantified using Qubit 2.0 fluorometer (Thermo Fisher), and 1 ug was digested separately with MspI, HindIII or HaeIII for 5TR cloning, and MspI, DpnII or HaeIII for 3TR cloning. The digested DNA was purified using QIAquick PCR Purification Kit. To prepare the individual LM-PCR linker mix, the equal volume of 50 uM linker common (long strand)(5' GACCCGGGAGATCTGAATTCAGTGGCACAGCAGTTAGGGGGCTGGTCG 3') and 50 uM restriction enzyme (RE)-digested end-specific linker (Blunt end linker /5Phos/CGACCAGCCC/3AmMO/ for HaeIII or CG end linker /5Phos/CGCGACCAGCCC/3AmMO/ for MspI or GATC end linker/5Phos/GATCCGACCAGCCC/3AmMO/ for DpnII or AGCT end linker/5Phos/AGCTCGACCAGCCC/3AmMO/ for HindIII were mixed in a PCR tube. The linker mix was then denatured and annealed by heating it to 95° C for 5 mins and cooling slowly to room temperature. One third of purified digested DNA was ligated with the individual LM-PCR linker mix using PfuTurbo Hotstart DNA Polymerase (Agilent Technologies). The ligation reaction was then digested with EcoRV (internal RE site of PB at 5TR direction) or MluI (internal RE site of PB at 3TR direction) to remove any linker that has ligated onto the 5TR or 3TR end of DNA, respectively. The remaining DNA containing MspI, HindIII and HaeIII linkers were pooled for 5TR sampling and the remaining DNA containing MspI, DpnII and HaeIII were pooled for 3TR processing. The pooled DNA was set up for PCR amplification using linker primer 1 (5' GACCCGGGAGATCTGAATTC3') and TR-specific biotinylated primers (LM_5TR_inver_A2: /5Biosg/AGATAATCATGCGTCATTTTGACTCAC or LM_3TR_inverA: /5Biosg/TCGATATACAGACCGATAAAACACATG). Biotinylated PCR products were pulled down using Dynabeads kilobaseBINDER kit at room temperature for 4 hours. Further nested PCR amplification was conducted using a primer that was specific for 5TR (LM_5TR_inver_B2: AGCGGCGACTGAGATGTCC) or 3TR (LM_3TR_inverB: ATGCGTCAATTTTACGCATGATTATC), and a Miseq sequencing index primer (Supplementary Table 5) containing individual sample barcode. The PCR products were tagged with modified P5 and P7 primers for sequencing.CITE-seq:after magnetic bead-based lineage depletion, prior to FACS sorting, each sample was stained with a pool of mouse CITE-Seq antibodies , and sample-specific hashing mixtures. Post staining and sorting, cells were run through the standard 10x Chromium (v3) protocol up until cDNA amplification, with the following modification: during cDNA PCR, 2 pmol of antibody-derived tag (ADT) additive primer (5'CCTTGGCACCCGAGAATTCC) and 1 pmol of hashtagging oligonucleotide (HTO) additive primer (5'GTGACTGGAGTTCAGACGTGTGCTC) were added to the cDNA amplification master mix. Post cDNA amplification, a 0.6X SPRI cleanup was performed to separate the cDNA fraction (on beads) from the smaller ADT and HTO fractions (in supernatant). The cDNA fraction was converted into a 3' tag gene expression library according to the 10x Genomics Single Cell Genomics Protocol (v3). Supernatant from cleanup was kept for ADT and HTO preparation. To the supernatant, another 1.4X SPRI was added to bring the total SPRI concentration to 2X. After washing the beads in 80% ethanol and eluting in water, a second round of 2X SPRI cleanup was performed to remove any residual primer carryover from the cDNA PCR. Post cleanup, eluate was taken into ADT PCR amplification (using TruSeq Small RNA RPIx primer (5'CAAGCAGAAGACGGCATACGAGXXXXXXXXGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA) and SI PCR primer (5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC)), and into hashtag amplification (using TruSeq D7xx (5'CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGC) and SI PCR primers). ). Libraries were pooled and sequenced on a 100 cycle Novaseq S1 flowcell. Additional protocol details can be found for CITE-Seq and cell hashing at www.cite-seq.com. RNA and DNA  libraries were prepared for sequencing using standard Illumina protocols RNA-Seq, Flip-Jump-Seq, CITE-Seq