Nextseq 75 cycle high output kit

Cells were resuspended at a final concentration of 120,000 cells/mL in PBS supplemented with 15% OptiPrep™ and libraries were prepared according to the inDrop protocol^{102 (link)} using the v3 barcoding configuration^{103 (link)}. Briefly, each cell sample was encapsulated in water-in-oil emulsions containing a barcoded polyacrylamide microgel, a lysis buffer, and reverse transcriptase mix. Each sample was collected in a single fraction containing approximately a 1000 cells and libraries were processed according to the protocol. The bead-bound barcode was released from the hydrogel using UV exposure and the droplets were incubated at 50 °C for 2 h followed by 70 °C for 20 min. The libraries were then processed for second-strand synthesis and amplification using in vitro transcription. After reverse-transcription, the libraries were amplified using limited-cycle indexing PCR. The final libraries were quality controlled using a BioAnalyzer HS kit (Agilent). Samples were pooled at equimolar ratios using both the BioAnalyzer HS and Qubit HS (ThermoFisher) metrics for size distribution and DNA concentration quantification. The libraries were sequenced on a Nextseq 75-cycle High Output kit (Illumina) in stand-alone mode using a 5% PhiX spike-in as an internal control.

CTCF ChIP was conducted using Millipore ChIP agarose kit (Millipore). 1 × 10⁶ cross-linked 416B cells were lysed and sonicated using a Covaris ultrasonicator. Sonicated chromatin was diluted using dilution buffer and 50 µL was removed as the 5% input control. 2 µL CTCF antibody (Supplementary Table 3) was added to 1 mL chromatin and incubated overnight at 4 °C. Decross-linking was done at 65 °C overnight. DNA was purified using phenol-chloroform-isoamylalcohol (25:24:1, Sigma) and enrichment was determined using qPCR (Supplementary Table 5). NEBNext Ultra II DNA Library Prep Kit (NEB) with 11 cycles of PCR was used to prepare sequencing libraries. CTCF ChIP libraries were sequenced using Illumina NextSeq high-output 75 cycle kit (paired-end).

RNA was isolated from 1 × 10³ to 2.5 × 10⁶ cells using QIAzol (Qiagen). Total RNA was extracted using miRNeasy Mini kit (Qiagen). RNA integrity was determined using a 4200 TapeStation RNA ScreenTape (Agilent). Ribosomal RNA was depleted from 2.5 µg total RNA per sample of undifferentiated mESC and 416B cells using the RiboMinus™ Eukaryote System v2 (Invitrogen). A poly A selection module (NEB) was used to extract poly A minus RNA and was eluted directly in First Strand Synthesis Reaction Buffer. cDNA was synthesized using the NEBNext Ultra directional library prep kit. Adapter ligation and 8–15 cycles of PCR were performed. Libraries were sequenced on Illumina NextSeq high-output 75 cycle kit (paired-end).